Dynamic Patterning of Maternal mRNAs in the
Early C. elegans Embryo
by
Jialing Li
B.A. University of California - Berkeley (2002)
Submitted to the Department of Physics
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June 2012
© Massachusetts Institute of Technology 2012. All rights reserved.
A uthor ...........................
... . . . . . . . I. . . . . .
... ...... .....
Department of Physics
April 6, 2012
......
Certified by ..........
L/
Alexander van Oudenaarden
Professor of Physics, Professor of Biology
Thesis Supervisor
X---
Accepted by .... ......
/
..
Krishna Rajagopal
Professor of Physics
Chairman, Associate Department Head for Education
2
Dynamic Patterning of Maternal mRNAs in the
Early C. elegans Embryo
by
Jialing Li
Submitted to the Department of Physics
on April 6, 2012, in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
Abstract
Asymmetric segregation of maternally-encoded proteins is essential to cell fate determination during early cell divisions of the Caenorhabditiselegans (C. elegans) embryo,
but little is known about the patterning of maternal transcripts inside somatic lineages. In the first Chapter of this thesis, by detecting individual mRNA molecules
in situ, we measured the densities of the two maternal mRNAs pie-1 and nos-2 in
non-germline cells. We find that nos-2 mRNA degrades at a constant rate in all somatic lineages, starting approximately 1 cell-cycle after each lineage separated from
the germline, consistent with a model in which the germline protects maternal mRNAs from degradation. In contrast, the degradation of pie-1 mRNAs in one somatic
lineage, AB, takes place at a rate slower than that of the other lineages, leading to an
accumulation of that transcript. We further show that the 3' untranslated (UTR) region of the pie-1 transcript at least partly encodes the AB-specific degradation delay.
Our results indicate that embryos actively control maternal mRNA distributions in
somatic lineages via regulated degradation, providing another potential mechanism
for lineage specification.
The evolutionary fate of an allele ordinarily depends on its contribution to host
fitness. Occasionally, however, genetic elements arise that are able to gain a transmission advantage while simultaneously imposing a fitness cost on their hosts. Seidel
et al. previously discovered one such element in C. elegans that gains a transmission
advantage through a combination of paternal-effect killing and zygotic self-rescue. In
the second Chapter of this thesis we demonstrate that this element is composed of a
sperm-delivered toxin, peel-1, and an embryo-expressed antidote, zeel-1. peel-1 and
zeel-1 are located adjacent to one another in the genome and co-occur in an insertion/deletion polymorphism. peel-1 encodes a novel four-pass transmembrane protein
that is expressed in sperm and delivered to the embryo via specialized, sperm-specific
vesicles. In the absence of zeel-1, sperm-delivered PEEL-1 causes lethal defects in muscle and epidermal tissue at the two-fold stage of embryogenesis. zeel-1 is expressed
transiently in the embryo and encodes a novel six-pass transmembrane domain fused
to a domain with sequence similarity to zyg-11, a substrate-recognition subunit of an
3
E3 ubiquitin ligase. zeel-1 appears to have arisen recently, during an expansion of
the zyg-11 family, and the transmembrane domain of zeel-1 is required and partially
sufficient for antidote activity. Although PEEL-1 and ZEEL-1 normally function in
embryos, these proteins can act at other stages as well. When expressed ectopically
in adults, PEEL-1 kills a variety of cell types, and ectopic expression of ZEEL-1 rescues these effects. Our results demonstrate that the tight physical linkage between
two novel transmembrane proteins has facilitated their co-evolution into an element
capable of promoting its own transmission to the detriment of the rest of the genome.
The Apical Epidermal Ridge (AER) in vertebrates is essential to the outgrowth
of a growing limb bud. Induction and maintenance of the AER reply heavily on
the coordination and signaling between two surrounding cell types: ectodermal and
mesenchymal cells. In morphogenesis during embryonic development, a process called
the epithelial-mesenchymal transition (EMT) occurs to transform epithelial cells into
mesenchymal cells for increased cell mobility and decreased cell adhesion. To check
whether the AER, which originated from the ectodermal layer, undergoes EMT for
enhanced cell motility and invasiveness at an early stage of the limb outgrowth, we
examined expression of biomarkers of the epithelial and mesenchymal cell types in
the AER of a mouse forelimb at embryonic day 10.5 in Chapter three of this thesis.
We also customized correlation-based image registration algorithm to perform image
stitching for more direct visualization of a big field of tissue sample. We found
that the AER surprisingly expresses both the epithelial marker and the mesenchymal
marker, unlike a normal non-transitioning epithelial cell or a cell undergoing EMT.
Our finding serves as a basis for potential future cell isolation experiments to further
look into cell type switching of the AER and its interaction with the surrounding
ectodermal and mesenchymal cells.
Thesis Supervisor: Alexander van Oudenaarden
Title: Professor of Physics, Professor of Biology
4
Acknowledgments
It is a pleasure to thank those who made this thesis possible. First and foremost, I am
grateful to my thesis adviser, Alexander van Oudenaarden, whose support, guidance,
patience, encouragement and funding are indispensable for me to develop and finish
the thesis project. I am also indebted to many of my colleagues, Arjun Raj, Bernardo
Pando, John Tsang, Qiong Yang and Dale Muzzey, to introduce me to experimental
biology and image processing, and Rui Zhen Tan, Mei Lyn Ong, Annalisa M Pawlosky
and Jan Phlipp Junker, to keep my PhD experience fun and stimulating.
I also
want to thank the Physics Department at MIT and all the female members of the
van Oudenaarden lab, especially Anna Lyubimova, and the Administrative Assistant,
Monica Wolf, for being extra supportive in my decision of balancing career and family.
Last but not the least, I owe my deepest gratitude to my family. Thanks to my parents
for raising me with a love of maths, science and problem-solving, and supporting me in
all my pursuits. Thanks to my husband, a fellow PhD student in theoretical Physics,
for staying motivational and accompanying me hurdling through grad school.
5
6
Contents
1
Dynamic Patterning of Maternal mRNAs in the Early C. elegans
9
Embryo
1.1
1.2
1.3
2
9
Introduction . . . . . . . . . . . . . . . . . . . .
1.1.1
The source of patterning in development
. . . . .
10
1.1.2
Founder cells and asymmetric divisions
. . . . .
11
1.1.3
The maternal-zygotic transition . . . . .
. . . . .
12
Experiments . . . . . . . . . . . . . . . . . . . .
. . . . .
13
1.2.1
Results . . . . . . . . . . . . . . . . . . .
. . . . .
14
1.2.2
Materials and Methods . . . . . . . . . .
20
. . . . .
Discussion . . . . . . . . . . . . . . . . . . . . .
1.3.1
RNA-specific modulation of degradation
1.3.2
Biological significance . . . . . . . . . . .
23
24
. . . . .
25
A novel sperm-delivered toxin causes late-stage embryo lethality and
27
transmission ratio distortion in C. elegans
2.1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
27
2.2
Experim ents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
2.2.1
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29
2.2.2
Materials and Methods . . . . . . . . . . . . . . . . . . . . . .
62
D iscussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
79
2.3.1
Sperm and early embryos may be protected from PEEL-1 . . .
79
2.3.2
Possible mechanisms of PEEL-1 toxicity and ZEEL-1-mediated
2.3
rescue ......
.......
...........
7
.........
... .
81
2.3.3
Comparison with other genetic elements causing transmission
ratio distortion
2.3.4
2.4
3
82
Sheltering of the peel-1/zeel-1 element by near perpetual hom ozygosity
2.3.5
. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evolutionary origins of the peel-1 /zeel-1 element
Acknowledgements
83
. . . . . . .
84
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
85
E- and N-cadherin in the developing mouse limb
87
3.1
87
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1
Cadherin switch and Epithelial-mesenchymal transition (EMT)
in development
3.2
. . . . . . . . . . . . . . . . . . . . . . . . . .
88
Preliminary Results . . . . . . . . . . . . . . . . . . . . . . . . . . . .
88
3.2.1
89
Image stitching method
. . . . . . . . . . . . . . . . . . . . .
A Supplementary Information
93
8
Chapter 1
Dynamic Patterning of Maternal
mRNAs in the Early C. elegans
Embryo
1.1
Introduction
C.elegans was first introduced as a model organism primarily for developmental biology and neurology researches by Sydney Brenner in 1965 [148]. It is a small, freeliving, non-parasitic, soil roundworm that is easy to grow and to maintain in a laboratory environment. Mutant strains can be easily frozen down and thawed up, and it
only takes a handful of animals to re-spawn a new colony. The major advantages of
using C. elegans for biology experiments are its short life-cycle and large progeny size,
which allow fast experimental turnaround and speed up the process of data collection.
Admittedly, C.elegans is less closely-related to humans compared to higher model organisms such as mice. And yet, its anatomical and developmental simplicity allows
us to begin to untangle the mysteries in the organization of multicellular organisms
[19].
To a physicist, biological processes such as embryogenesis and regeneration are
puzzling.
These processes are great examples of self-organizing systems in which
9
ordered patterns and structures arise from a seemingly uniform field of ancestor cells,
often in response to some external cue and may involve feedbacks to amplify an
initial bias.
Incidentally, the ideas of self-organization, patterning, and clustering
have become an almost ubiquitous theme in recent years, extending from the study
of protein interactions to the analysis of flocking behavior and social networks [48,
92, 97].
1.1.1
The source of patterning in development
The Turing pattern was proposed by the great mathematician Alan Turing in 1952
[135]. Turing wanted to show that combinations of known physical interactions are
sufficient to explain pattern formation in biological systems. In his reaction-diffusion
model, Turing demonstrated robust spatial patterns can develop autonomously in a
simple system composed of a short-range activator and a long-range inhibitor. A key
feature of Turing's model is that it does not require a pre-existing asymmetry and
therefore is truly a self-organizing system.
The periodicity and autonomous nature of the Turing pattern might sound promising for modeling biological systems where repetitive structures form with little periodic pre-patterning (for review, see [681). For example, a Turing-related model has
been suggested to be applicable for digit formation during vertebrate limb development [94]. Miura et al. recapitulated the appearance of extra digits in a polydactylous
mouse mutant with numerical simulations based on the Turing reaction-diffusion sys-
tem [89].
The reaction-diffusion model is quite powerful. However, in most cases of development a cell relies on the positional information in decision makings such as which
fate to adopt, whether to undergo programmed cell-death, etc. The question is where
this information comes from and how the cell processes that information.
In our current understanding, a cell could gather its positional information from
two potential sources. The obvious one is through morphogen diffusion and cell-cell
interactions, which enable a cell to physically sense its environment and act accordingly. Alternatively, a cell is capable of knowing innately where it is in terms of lin-
10
eage ancestry and developmental stage. It does so by maintaining certain genetic and
epigenetic markers, namely, cell fate determinants. Most cell ablation experiments
performed in C.elegans in the 1980's failed to perturb identities of the remaining cells,
suggesting cell fates might be largely intrinsically determined [128]. In the late 1980's
and the 1990's, blastomere isolation experiments together with genetic screens have
demonstrated cell-cell interactions also play a major role in cell-fate determination
in C.elegans embryogenesis [47, 60, 103, 108, 134]. In conclusion, the specification of
blastomere identity could depend on both intrinsic signals and external cues.
1.1.2
Founder cells and asymmetric divisions
Like most other multicellular organisms (not including the ones with the power of
asexual reproduction through regeneration), C.elegans begin its journey of development from one single cell. From fertilization to gastrulation, five somatic founder cells
are generated, through rounds of asymmetric cell divisions. These founder cells are
well-characterized in terms of which germ layer their progeny contribute to [127, 126].
At 2-cell stage, the first founder cell AB is born, and its descendants are mostly ectodermal. The EMS cell is born at 4-cell stage, and further divides into two founder
cells E and MS one cell-cycle later. The progeny of E cell makes up the endoderm
exclusively, while MS descendants belong primarily to the mesoderm layer.
Conceivably, asymmetric cell divisions serve as an ideal strategy for self-renewal
and differentiation of stem cells. Genetic screens in C.elegans have uncovered a family
of regulator proteins called Par proteins responsible for the initial polarity establishment (following an external cue, namely, the sperm entry site) and the asymmetric
partitioning of cell fate determinants during the first cell cycle [17, 36, 50, 56, 76, 91,
143]. Homologues of all-but-one Par proteins are found to exist and to be functionally
conserved in higher organisms [4, 27, 78].
11
1.1.3
The maternal-zygotic transition
In many animals, the mother expresses and deposits a significant fraction of mRNAs
into the oocyte before fertilization ([10], reviewed by [40]).
The presence of these
maternal transcripts allows the first few hours of embryogenesis to proceed without
transcription of the embryonic genome ([33, 34], reviewed by [130]) because maternally
supplied mRNAs encode proteins sufficient for establishing embryonic polarity and
initializing lineage specification [15, 50, 61, 110].
During the subsequent transition
from maternal to zygotic gene expression, the levels of many maternal mRNAs drop
significantly, allowing zygotic transcription to assume control of the developmental
program ([10], reviewed by [114]).
Because embryos lack control of maternal mRNA transcription, post-transcriptional
control of maternal mRNAs is crucial for normal developmental progression.
In
Drosophila melanogaster (D. melanogaster), for instance, maternal mRNAs display
marked spatiotemporal patterns. Examples include the localization of nanos and bicoid mRNAs to the anterior and posterior of the embryo, respectively, thus forming
the protein gradients that establish the anterior-posterior axis [124]. The dominant
mechanism for the localization of these mRNAs is the transport of the mRNAs, either
active or passive, to its final destination ([25], reviewed by [98]). Such transportationbased mechanisms are useful in the syncytial cytoplasm of the Drosophila embryo;
however, they would not be effective in the cellularized embryos in which the cellular
membranes would block mRNA transport.
In a cellularized context, the remaining option for spatiotemporal patterning of
maternal mRNAs is the regulation of degradation. Thus far, the evidence for this
mechanism arises from the observation of the protection of maternal mRNAs in the
germline of the developing organism [7, 32, 116, 147]. In C.elegans, in situ hybridization revealed that C. elegans maternal mRNAs fall into two primary classes based
on their germline-versus-soma distributions: Class I defines transcripts with uniform
densities throughout the embryo and relatively constant whole-embryo levels from fertilization through gastrulation and beyond (mostly housekeeping genes), while Class
12
II maternal mRNAs showed strong enrichment in the germline compared to the soma,
with overall levels in the soma dropping significantly by gastrulation [10, 118]. The
lack of evidence for cell-specific patterning of maternal mRNA levels in the soma,
however, has led to the belief that, unlike the syncytial fly embryo, much of the
early patterning of the somatic worm embryo relies largely on translational control
and protein localization (reviewed by [38, 111]). Indeed, there is substantial evidence
for this view; for instance, the 3' UTR regulates the translation of several maternal
genes important for the patterning of the early embryo, such as pal-1, glp-1 and nos2 [29, 37, 57, 87]. The possibility that maternal mRNA levels in the soma could be
subject to lineage-specific regulation, however, remains unexplored, largely due to the
inability to systematically measure the low levels of the mRNAs involved.
1.2
Experiments
In this thesis, we chose to survey the distributions of two maternally deposited mRNAs
in different somatic lineages of the early C. elegans embryo. C. elegans is a powerful
tool for studying processes related to embryogenesis thanks to its invariance in cell
identities and positioning, which greatly facilitates experimental reproducibility. In
addition, the complete lineage tree and timing of cell divisions have been previously
recorded, which makes staging the embryos a simple task [5, 126].
We have studied the regulation of the stability of two maternal RNAs using a
single-molecule mRNA counting method based on fluorescence in situ hybridization.
Our data shows that in each of the somatic lineages the degradation of nos-2 mRNA
begins one cell-cycle after their separation from the germline, with rates being constant in time and very similar among the AB, EMS and C lineages. The extent of
depletion of nos-2 mRNA is always higher in AB than in EMS, due to the earlier
onset of degradation in AB, an effect we have called the germline protection model.
We also examined the degradation of pie-1 mRNA, revealing somatic preservation of
it in the AB lineage until the 15-cell stage, strong enough to cause a faster depletion
of it in EMS than in AB. This differential regulation of pie-1 and nos-2 degradation
13
results in starkly different enrichment patterns for those two maternal mRNAs in the
AB versus EMS lineages. Subsequent experiments demonstrated that the 3' UTRs
contain important regulatory elements responsible for generating the different spatiotemporal patterns of pie-1 and nos-2 mRNA levels in the soma.
1.2.1
Results
We measured RNA abundance by computing combined RNA densities of AB descendants or EMS descendants over time. We observed that both pie-1 and nos-2
transcripts degraded much more quickly in somatic lineages than in the germline
throughout the cell stages examined (from 1 cell to 28 cells; examples of an early and
a late stage embryo shown in Fig. 1-1 A and B), consistent with previous studies of
Class II maternal mRNAs [29, 118, 131]. In contrast with previous studies, however,
we found that between the 1 and 4 cell stages, the combined number of mRNAs in the
P 2 (Pi) and EMS (AB) cells was similar to those in their immediate ancestor, the Pi
(PO) cell, whereas the total number of mRNAs in the ABa and ABp were lower than
their immediate ancestor (AB) (Fig. 1-1 C). In other words, the percent decrease in
maternal mRNA counts was significantly lower in the germ cells immediate daughters
than in other somatic cells. This suggests that the onset of degradation in the somatic
cells begins one cell cycle after the somatic cell divides away from the germline P cell.
This could be due to the presence of mRNA protection machinery (such as P granules)
inherited from the mother P cell or slow maturation of degradation machinery in the
newly born somatic cell [12, 44]. Based on our data, we propose a germline protection model for somatically degraded maternal mRNA in which mRNA degradation
is stalled until one cell cycle after each somatic lineage separates from the P-cell and
proceeds at a constant rate thereafter (Fig. 1-2 A and B); in other words, maternal
mRNAs in the direct descendants of P cells are temporarily protected. This model
serves as a null-model of what one might expect to see in the absence of any lineage
specific regulation of maternal transcripts.
The germline protection model further predicts the differences in mRNA density
should persist at later stages of embryogenesis; in particular, the mRNA density in
14
C
pie-1
nos-2
5.8% decrease
4.5% decrease
1-cell
2-cell
9.2% decrease
13.7% decrease
3.5% decrease:
-
15.6% decrease
4-cell
0
2000
4000
0
2000
4000
6000
mRNA Counts
N germ lineage
U
somatic lineages
Figure 1-1: Class II maternal mRNAs degrade faster in the soma than in
germline. (A and B) Visualization of pie-i transcript levels in a 4-cell embryo (A)
and in a 15-cell embryo undergoing a new round of cell divisions (B) with DAPI
counterstaining for the nuclei (blue) in a transgenic line with GFP cell boundary
label (green). Shown in magenta are diffraction-limited spots representing individual
transcripts. The dashed blue curve outlines the AB lineage in (B). Shown here are
maximum projections of three planes (with a 0.3 pm interplanar space) out of thirty
planes total per embryo. Scale bars are 5 pm long. (C) Counts of pie-i and nos-2
mRNAs at 1 (n=13), 2 (n=8), and 4 (n=12) cell stages in GFP:PH embryos. Orange
bars represent the germline P cells, and the blue and green bars indicate the somatic,
AB, and EMS lineages respectively. Error bars indicate 95% confidence intervals for
the sample means. Both pie-1 and nos-2 transcripts were degraded more rapidly in
the somatic lineage (from AB to ABa plus ABp) than in the germ lineage (from P1
to P2 plus EMS); two-sample t-test: p = 0.0096 for pie-1, p = 0.00032 for nos-2.
15
B
A
germline protection model
k
Po
P2
AB
5
C
CU)~
P3
P4
C
0.4
E
pie-1
mRNA
U)
nos-2
mRNA
e
C
e
0
e
S
0
0
2:1
.F0.
0
eS
o0
Number
of Cells
12
13
14
15
24-28
EMS-to-AB
volume ratio
0.32 ± 0.05
0.36 ± 0.07
0.37 ± 0.04
0.39 ± 0.04
0.36 ± 0.07
0
0
0.4
0.2
3
Density in AB Lineage (pm0 )
Figure 1-2: Concentration of pie-1 mRNA violates the germline protection
model with a less-than-1 EMS-to-AB ratio. (A) Theoretical degradation curves
of maternal mRNAs in the somatic lineages based on germline protection model,
predicting that the later onset of degradation would lead to a higher concentration
of maternal mRNAs in EMS than in AB. (B) Lineage tree from 1 to 24 cell stages
with colors corresponding to the lines in (A). (C) Densities of pie-i (blue) and nos-2
(red) mRNAs in EMS versus in AB of wildtype (N2) embryos over a developmental
window from 12 to 28 cell stages. Lighter spots correspond to later stages as indicated
in the chart on the right. Listed on the right are the mean volume ratios between the
EMS cells and the AB cells, ± errors in the volume ratio representing 95% confidence
intervals for the sample means.
16
the EMS lineage should always be higher than in the AB lineage because EMS divides
away from the P-cell one cell cycle later. To test this prediction, we measured the
abundance of both nos-2 and pie-1 in the different lineages in embryos containing
between 12 and 28 cells. We measured this abundance by computing the mRNA
density rather than the total mRNA count in a lineage because the total count is
rather sensitive to minor fluctuations in cell boundary identification during manual
segmentation. Given that the volume ratio of EMS descendants to AB descendants
is relatively constant during this developmental window (Fig.
1-2 C), the use of
density rather than total count does not lead to systematic errors in our analysis. We
found that nos-2 mRNA levels were consistent with the germline protection model,
with a substantially higher density of mRNAs present in the EMS lineage than in
the AB lineage at virtually all cell stages (Fig. 1-2 C). Surprisingly, though, pie-i
mRNA levels exhibited exactly the opposite behavior, with the mRNA density in
the AB lineage being consistently higher than in the EMS lineage, showing that the
degradation of pie-1 is subject to lineage specific regulation.
The high density of pie-1 in the AB lineage as compared to the EMS lineage
could be due to either (or both) of two factors: a mRNA specific degradation of pie-i
in the EMS lineage, or a mRNA specific block of degradation of pie-i in the AB
lineage. To distinguish between these two possibilities, we quantified the degradation
dynamics of both mRNAs in the AB, EMS and C lineages. In order to measure the age
of our embryos, we used high resolution temporal lineage information [5] to convert
number of cells into time elapsed since the first cell division. As previously mentioned,
nos-2 exhibits a degradation pattern consistent with the germline protection model.
Notably, the density of nos-2 mRNA follows an exponential curve a short time after
the lineages separate from the P cell (Fig. 1-3 A), with the degradation rate computed
from that curve being similar among the different lineages (Fig. 1-3 B). pie-1 mRNA
densities likewise follow a simple exponential in the EMS and C lineages, but the
density curve in the AB lineage shows a biphasic behavior, in which degradation
before the 15 cell stage (-74 min after first cell division) is almost three-fold slower
than in the EMS and C lineages, although degradation in the AB lineage after that
17
A
EMS
AB
C
0.6
0.3
Early
Late
0
0.8
E
C,)
,
N
*44
"V
0.4
.--D
0
50
-T
70
90
50
70
90
50
70
90
Time After First Cell Division (min)
B
pie-1
70.16
nos-2
-
C
C
I
Late
0
1~
0.0
T
CD
T
CD
I
z
E
0-
AB
EMS
C
AB
EMS
C
Figure 1-3: Degradation of pie-1 transcripts in the AB lineage proceeds at
a slower rate prior to 15 cell stage (~74 min after first cell division) than
thereafter. (A) Experimentally measured degradation curves of pie-i and nos-2
mRNAs in the various somatic lineages, with the estimated germline degradation
curves in orange in the background. The time points used in the plots correspond
to the following cell stages: 6, 8, 12, 13, 14, 15, 24 and 28. Error bars indicate
95% confidence intervals for the sample means. (B) Exponentially fitted rates of
pie-i and nos-2 degradation among different lineages found by the curve fitting in
(A). Error bars indicate 95% confidence intervals obtained by bootstrapping (see
Statistical Methods).
18
C
gfp:h2b:nos-23'UTR
1.8
D - gfp:h2b:pie-1 3'UTR
CU
-+) nos-2
1e-
13
(
-
-
14p
C)
0C:
c:
E0.6-
E
Q=
12
13
14
Number of Cells
15
Figure 1-4: Both pie-1 and nos-2 3' UTR can direct the mRNA degradation
pattern of exogenous gfp:h2b transcripts. (A and B) Simultaneous visualization of gfp:h2b transcripts with either pie-i or nos-2 3' UTR and the corresponding
endogenous mRNA with DAPI counter staining for the nuclei, in 15 cell stage transgenic embryos of N2 background. Shown here are maximum projections of three
planes (with a 0.3 pm interplanar space) out of thirty planes total per embryo. Scale
bars are 5 pm long. (C) EMS-versus-AB density ratios of the following transcripts
in embryos staged from 12 to 15 cells: gfp:h2b:pie-1 3' UTR, gfp:h2b:nos-2 3' UTR,
endogenous pie-i and nos-2. Error bars indicate 95% confidence intervals for the
sample means.
19
time is again similar to that observed in the other lineages. These data point to a
specific regulation of pie-1 mRNA degradation in which degradation is selectively
blocked in the AB lineage during a specific phase of development.
Regulation of maternal mRNAs is often encoded in the 3' UTR of the transcript
[1, 8, 33]. With this in mind, we set out to test whether the 3' UTR plays a role in encoding the regulation of pie-1 mRNA stability. To this end, we measured mRNA distributions in two strains, each containing a GFP-fused histone protein gene (gfp:h2b)
driven by the pie-1 promoter with either the pie-1 or the nos-2 3' UTR (Fig. 1-4
A and B). We found that the gfp:h2b transcripts with the nos-2 3' UTR follow the
germline protection model, while those with pie-1 3' UTR are more abundant in the
AB lineage than in the EMS lineage in embryos containing between 12 and 15 cells
(Fig. 1-4 C), qualitatively mimicking the behavior of the endogenous transcripts.
The observed differential degradation in the gfp:h2b transcripts is not as strong as
in the endogenous transcripts, suggesting the existence of additional regulatory elements in the coding portions of the transcripts. Nevertheless, our results show that
the dynamics of pie-1 and nos-2 mRNA degradation is specific to those transcripts
and is regulated via the 3' UTR.
In Zebrafish the clearance of maternal transcripts is accelerated by the MiR-430
microRNA [45]. In C. elegans, the loss of mir-35 or mir-51 microRNA family leads
to embryonic lethality. We have examined the pie-1 and nos-2 transcript levels in
mutant strains lacking microRNAs from either the mir-35 or the mir-51 families and
failed to observe any significant changes from the wildtype distributions.
1.2.2
Materials and Methods
C. elegans strains and culture methods
We cultured C. elegans at 20C by following standard procedures and used the Bristol N2 as the wildtype strain [20].
We obtained the GFP cell-membrane labeling
strain (OD58(Ppie-1 gfp:ph(PLC161))) from the Oegema lab [3]. The mutant strains
lacking mir-35-41(nDF50) and mir-51(n4473) were gifts from the Horvitz lab [2].
20
Other transgenic strains used in this study were gifts from the Seydoux lab [29, 87]:
JH2379(Ppie-1gfp:h2b:pie-13' UTR), JH2000(Ppie-1gfp:h2b:nos-23' UTR).
Single Molecule Fluorescence in situ Hybridization (FISH)
We carried out three-color single-molecule FISH procedures on C. elegans embryos as
previously described [105], for simultaneous visualization of the two out of the three
RNA species of interest (pie-1, nos-2 and gfp:h2b) together with EMS lineage-specific
transcripts (end-3 and med-1,2 combined).
We conjugated each set of oligonu-
cleotides targeting a unique transcript species including pie-1, nos-2 and gfp:h2b to
either tetramethylrhodamine (TMR), Cy5 or Alexa594. end-3 and med-1,2 probes
were generated previously [104]. Signals from GFP declined but still stayed well above
detection level after performing FISH. We gently squashed the embryos to -9 micron
thick before imaging. The oligonucleotide sequences of all the probes used in this
study are listed in the supplementary material.
Counting mRNAs in individual cells and lineages
We identified and localized diffraction-limited spots representing single mRNA molecules
using MATLAB software developed by Raj et al. [105] with slight modifications. The
original software package utilizes a Mexican hat (Laplacian of Gaussian) filter for signal enhancement, in addition to manual thresholding for eliminating background noise
and for positive identification of mRNA spots. A Gaussian filter smoothes out background noise and a Laplacian filter takes the spatial derivative of an image and detects
rapid intensity changes between signal and background. Because an input image is
composed of discrete pixels, we can apply both the Gaussian and the Laplacian filters
using standard convolution methods. To speed up the image processing time, one
can convolve the Gaussian filter with the Laplacian filter beforehand and apply the
resulting hybrid filter to the input image altogether, given that the convolution operation is associative. The original package developed by Raj et al. [105] works nicely
for resolving adjacent mRNA spots with not too small spatial separations; however,
resolving diffraction-limited spots that overlap depends critically on the threshold
21
level and thus becomes a source of couting error. A single diffraction-limited spot
has a Gaussian spread under widefield fluorescent microscope. When two diffractionlimited spots are within close proximity, their Gaussian spreads start to overlap. In
the case where intensities of two overlapping Gaussian tails adds to be well above
the threshold level, two spots will be mistakenly counted as one. To circumvent this
issue, we identified regional maxima of the Laplacian filtered images which indicate
the center locations of individual spots. Isolation of these maxima allowed us to reset
the intensity in overlapping region between spots down to background level and to
resolve overlapping mRNA spots regardless of the threshold chosen.
We manually segmented individual cells in embryos up to 4 cell stage based on
the GFP:PH cell membrane signal. For stages beyond 4 cell and until 28 cell, we sequentially identified the different lineages (P, D, C, EMS and AB, if present) based on
cell division time, sizes and locations of the nuclei with 4',6-diamidino-2-phenylindole
(DAPI) staining, together with mRNA density information. The germline (P) lineage was easily recognizable by its posterior end location and the hallmark highest
concentration of maternal RNAs, nos-2 and pie-1. The C cell and its descendents
sit adjacently to P and their cytoplasm contain the highest density of nos-2 mRNAs
among all the somatic lineages until the birth of D at 24 cell stage. In addition, we
labeled end-3/med-1,2 mRNA using FISH for marking the cytoplasm of EMS descendent cells. At last, after marking all the other lineages in an embryo, we tagged
what remained unmarked to be the AB lineage automatically using MATLAB. We
then measured the volumes of all lineages, with their shapes changing continuously
throughout the 30 planes of images taken for each embryo, an 0.3 micron interplanar
distance and a conversion factor of 8 camera pixels per micron. We then counted the
mRNA spots inside each lineage to get the density.
Statistical methods
We assumed the mRNA degradation rates are normally distributed among the population and performed a two-sample t-test for the null hypothesis of 2-to-4 cell stage
embryos having equal degradation rates of pie-1 and nos-2 in soma and germline (ver22
sus the alternative hypothesis of the rates being higher in soma than in germline).
With one exception, we fitted each of the experimentally measured degradation curves
of pie-1 and nos-2 mRNAs in the various somatic lineages to a single exponential decay curve with a fixed onset time consistent with the germline protection model using
nonlinear least-squares. The exception is the degradation curve of pie-1 mRNA in
the AB lineage, to which we fitted two consecutive exponentially decaying curves, by
allowing the onset time of the second exponential to be an additional free parameter.
To obtain error estimates on degradation rates obtained from the exponential fits, we
performed the same least square fitting on 5000 bootstrapped data sets (simulated
data sets randomly resampled from the original data).
1.3
Discussion
We observed interesting dynamics reflecting lineage ancestry in both maternal RNA
species. For nos-2, its densities within the somatic lineages follow essentially the same
dynamics after each asymmetric cell division. After the birth of each somatic lineage,
the nos-2 density within that lineage starts out high and rapidly decreases after one
cell-cycle.
Therefore, the rank of nos-2 densities from the lowest to the highest,
at stages prior to gastrulation, faithfully represents the birth order of the somatic
lineages. The density curves of pie-1, on the other hand, show two distinct behaviors.
In the AB lineage, the dynamic of pie-1 is bi-phasic, with a slower initial decay and a
more rapid subsequent drop. In the EMS and C lineages, pie-1 behaves pretty much
the same as nos-2 does. Unlike nos-2, however, the density of pie-1 stayed higher in
the earlier born AB lineage than in the later born EMS lineage from 8-cell to 24-cellstage. Our result has suggested that the developing embryo intricately regulates the
degradation of maternal mRNAs, so as to generate differential patterning of different
mRNA species.
23
1.3.1
RNA-specific modulation of degradation
We can approximate the degradation of mRNAs as a simple process in which the
degradation machinery (DM) binds and removes the target mRNA. (In the case of
the germline lineage,the P-granules protect maternal RNAs from degradation.) The
resulting mRNA density is an exponential function with a negative decay rate proportional to both the concentration of DM and the RNA-DM binding rate constant.
The non-linearity in this transformation requires a fine tuning of DM concentrations
and the binding constant.
In other words, a small alternation in the binding effi-
ciency between the mRNA species and its degradation machinery could cause a huge
disparity in the mRNA distribution over time.
We have shown pie-1 and nos-2 mRNAs decay with very different kinetics through
direct comparison. Moreover, we measured a rapid shift in the decay rate of pie-1
transcripts at a specific developmental stage. Our findings, together with suggestions
from previous in situ studies of other maternal transcripts such as pos-1, pal-1 and glp1 [37, 57, 118, 119, 129], imply the existence of mRNA-specific controls of degradation.
Whether these controls are direct modulations or merely secondary consequences of
regulated translation is an interesting topic for future research. We cannot rule out
the latter as a possibility.
If indeed our findings reflect a direct modulation in the decay process of pie-1
mRNA, what could have caused the initial slowdown of pie-1 mRNA degradation in
AB? Perhaps the degradation of pie-1 transcripts is actively blocked in early developmental stages. Alternatively, pie-1 mRNA decay might require a special zygotic
element. Bashirullah et al. and others previously reported the existence of distinct
maternal and zygotic decay machineries in Drosophila and that distinct zygotic decay activities can target different maternal transcripts [8, 31]. Given our result, we
speculate that pie-1 transcripts might be targeted by a zygotic decay activity which
switches on late. During the maternal-to-zygotic transition, differential timing of activation of different zygotically controlled decay machineries could be a driving force
of differential patterning of distinct maternal transcripts.
24
One fundamental question about C. elegans development is how the various somatic lineages are derived from the same (germline) lineage through sequential asymmetric cell divisions and yet segregate into distinct fates. The early C. elegans embryo
contains many strictly maternal (maternal but not embryonic) transcripts, including
those of pie-1 and nos-2, which can only be degraded but not newly synthesized [10].
The lack of cell specific expression presents a potential problem for using maternal
mRNA to specify cell fates because in the absence of active intercellular transport of
maternal transcripts (a strategy employed in the Drosophila syncytium, [52]), regulation of degradation is the only remaining option. Our results show that intricate
regulation of degradation encoded in the 3' UTR can provide exactly such a mechanism for generating cell-specific maternal mRNA abundances, in this case, resulting
in unique spatiotemporal patterns of pie-1 and nos-2 transcripts. By programming
degradation rates in a transcript-specific manner, several maternal factors can act coordinately in generating stage- and lineage-specific expression patterns during early
embryogenesis.
1.3.2
Biological significance
We were surprised to find preservation of pie-1 maternal RNA in the AB lineage,
because previous studies primarily demonstrated that the PIE-1 protein, a key regulator for germline differentiation, segregates preferentially to the P lineage and is
actively degraded in the soma throughout early development [86, 132]. No patterning
of PIE-1 protein has been observed in the soma to this date, likely because of the
lower sensitivity of GFP tagged proteins and immunostaining methods. The single
molecule FISH method we employed here detects changes in the absolute mRNA
counts [105], providing us with the sensitivity to quantitatively measure differences
in mRNA densities.
What possible functions might the regulated stability of pie-1 mRNA serve? Researchers have shown that the post-translational control of the pie-1 gene mainly
serves to concentrate PIE-1 protein in the germline, so as to prevent the germline
from adopting somatic fate [119]. In particular, PIE-1 represses SKN-1 in the germ
25
lineage and release of this repression will cause the germline to take on the EMS
identity [15, 16, 84, 86]. We speculate that the spatiotemporal regulation of maternal
pie-i transcripts, i.e. to retain higher level of pie-1 mRNA in AB than EMS, helps
reinforce the differences between AB and EMS by acting together with other somatic
skn-1 repressor genes to maintain a low level of SKN-1 protein in AB. Further work
is required to show the generality of such mechanisms for regulation of cell fate via
regulated degradation of maternal transcripts.
26
Chapter 2
A novel sperm-delivered toxin
causes late-stage embryo lethality
and transmission ratio distortion in
C. elegans
2.1
Introduction
The evolutionary fate of an allele ordinarily depends on the reproductive fitness of the
organisms carrying it. In some cases, however, alleles are able to increase their representation in future generations while being neutral or detrimental to the fitness of
their bearers. These elements, sometimes termed selfish or parasitic genes, influence
transmission probabilities in a variety of ways. Some self-replicate and insert themselves into new genomic locations (e.g. transposons) [59]. Others act during meiosis
to preferentially segregate into the oocyte [30, 41, 99] or to reduce the viability of
sperm or spores inheriting alternate alleles [63, 70, 9, 136]. Still others act at the level
of the zygote to destroy progeny not inheriting them. Medea-factors in Tribolium destroy non-carrier animals through a combination of maternal-effect killing and zygotic
self-rescue [79]. An analogous phenomenon occurs in organisms infected by the ma-
27
ternally transmitted bacteria, Wolbachia or Cardinium [117, 144, 58], which modify
the sperm of infected males to cause lethal defects when paired with the oocytes of
uninfected females.
Previously, Seidel et al. discovered a nuclear-encoded element in C. elegans that
kills non-carrier animals in a novel way [115]. This element, referred to as the peel1 /zeel-1 element, is polymorphic within the species, and when animals carrying the
peel-1/zeel-1 element are crossed to animals lacking it, the peel-1/zeel-1 element
acts in the F1 heterozygote via paternal effect to kill F2 or backcross embryos not
inheriting it.
This lethality acts independently of maternal genotype and causes
the peel-1/zeel-1 element to become over-represented among the viable progeny of
heterozygous sires, even while incurring a substantial fitness cost to these animals.
Paternal-effect loci are extremely rare in all of biology [21, 42], and the observed
combination of nuclear-encoded, paternal-effect killing and zygotic self-rescue is unprecedented. In C. elegans, moreover, a paternal-effect locus whose effects can be rescued zygotically is mechanistically surprising because in this species, sperm-supplied
factors are thought to act only during fertilization and first cleavage [77], whereas
zygotic transcription does not begin until the four-cell stage [118].
Although the peel-1 /zeel-1 element is capable of promoting its own transmission,
it rarely has the opportunity to do so in natural populations. C. elegans is an androdioecious species that reproduces primarily through self-fertilizing hermaphrodites.
Because inbreeding is high [6], the peel-i/zeel-1 element normally exists in the homozygous state, where no opportunity for self-promotion exists.
High inbreeding notwithstanding, out-crossing events in C. elegans between hermaphrodites
and males do occur, albeit rarely [6, 28, 109]. And because the peel-1 /zeel-1 element
is globally distributed and confers no apparent fitness disadvantage in the homozygous state [115], this element is expected to drive itself to fixation faster than a
neutrally evolving locus. Consistent with this prediction, in laboratory populations
where out-crossing is forced, the peel-1/zeel-1 element fixes rapidly [115]. In natural populations, however, the peel-1/zeel-1 element has remained polymorphic for an
estimated 8 million generations [115], much longer than expected under neutrality.
28
One likely explanation for this paradox is that the peel-1 /zeel-1
element is tightly
linked to a polymorphism maintained by balancing selection, and the tightness of this
linkage maintains the peel-1/zeel-1 element in the polymorphic state [115].
Given the unusual genetics of the peel-1/zeel-1 element, we sought to understand
its mechanism of action. Seidel et al. previously identified one component of the peel1/zeel-1 element as the gene zeel-1 (Y39G1OAR.5), which acts zygotically to rescue
Here we demonstrate that zeel-1 is fully separable
the paternal-effect killing [115].
from the paternal-effect killing, and that this killing activity is encoded by a second
gene, peel-1 (Y39G10AR.25). We show that PEEL-i acts as a sperm-supplied toxin,
and ZEEL-1 an embryo-expressed antidote. We characterize the developmental defects caused by sperm-supplied PEEL-1, and we report a dose-dependent relationship
between the severity of these defects and the quantity of PEEL-i delivered to the
embryo.
We analyze the phylogenetic origins and functionality of each domain of
zeel-1, and we test the tissue-autonomy of zeel-1 rescue. Finally, in order to determine whether peel-1 and zeel-1 can function outside of embryogenesis, we express
both genes ectopically in adults.
2.2
2.2.1
Experiments
Results
peel-1 and zeel-1 are genetically distinct
The genetics of the peel-1/zeel-1 element are consistent with it being composed of
two interacting loci: a dominant-lethal, paternal-effect toxin, peel-1, and a zygotically
acting antidote, zeel-1 [115].
The activities of peel-1 and zeel-1 are present in the
reference strain, Bristol (N2), and in approximately two-thirds of wild isolates [115].
These strains are said to carry the peel-1/zeel-1 element. The commonly used wild
strain, collected from Hawaii (CB4856), and all but two of the additional wild strains
lack the activities of both peel-1 and zeel-1 [115].
The two remaining strains, one
collected from Germany (MY19) and one collected from Utah (EG4348), exhibit the
29
activity of zeel-1 but are unable to induce the paternal-effect killing [115], (Figure
2-1).
Seidel et al. previously mapped the peel-1/zeel-i element in the Bristol strain
to a 62 kb interval on the left arm of chromosome I [115]. Within this interval, we
identified a single gene capable of providing antidote activity. This gene, which we
named zeel-1, encodes a 917-amino acid protein whose N-terminus is predicted to form
a six-pass transmembrane domain and whose C-terminus exhibits sequence similarity
to ZYG-11, a substrate-recognition subunit of an E3 ubiquitin ligase [65]. The Hawaii
strain carries a 19 kb deficiency (niDf9) spanning zeel-1, and this deficiency is shared
by all other wild isolates lacking the activities of both peel-1 and zeel-1 [115]. This
deficiency is not shared by wild strains carrying the peel-1/zeel-1 element, nor by
MY19 or EG4348 [115].
The phenotype of MY19 and EG4348 demonstrates that the zeel-1 gene is not
sufficient for peel-1 activity. Conversely, a deletion allele of zeel-1 in the Bristol background demonstrates that zeel-1 is also not required for it. This deletion, tm3419,
removes 221 base pairs spanning the start codon of zeel-1 (Figure 2-2 A). As expected, this deletion abolishes antidote activity (Figure 2-1 B); however, it does not
abolish the paternal-effect killing (Figure 2-1 B). zeel-1 is therefore genetically separable from a second, paternal-effect locus, peel-1. We note that as a consequence of
this separability, zeel-1 deletions in the Bristol and Hawaii backgrounds have opposite phenotypic effects: the niDf9 deficiency is perfectly viable, whereas the tm3419
deletion behaves as a conventional recessive-lethal mutation.
peel-1 encodes a novel, four-pass transmembrane protein of unknown function
In MY19 and EG4348, absence of peel-1 activity is tightly linked to the 62 kb peel1 interval [115], (Figure 2-1 C), suggesting that these strains carry loss-of-function
alleles of peel-1, rather than extra-genic suppressors. In addition, sequence analysis
of the peel-i interval in MY19 [115] and EG4348 (see Materials and Methods) indicates that absence of peel-1 activity in these strains is not caused by recombination
30
A
% Ernbryo lethality
Cross
(total)
EG4348/Bristol selfing
0% (275)
EG4348/Bristol
d
X Hawaii
0.7% (280)
EG4348/Hawaii selfing
0.7% (285)
EG4348/Hawaii r X Hawaii
0.3% (372)
Bristol/Hawaii selfing
23% (209)
Bristol/Hawaii
d
51.2% (242)
X Hawaii
B
% Embryo lethality
(total)
Expected Observed
Cross
zeel-1(tm34 19)
selfing
+
zeel-1I(+) peel- 1(+)
-25%
24.2% (1009)
zeel-1(tm3419) selfing
zeel-1 (A)peel-1(A)
-100%
99.4% (994)
100%
100% (881)
zeel-1(tm3419)
zeel-1(A) peel-1(a)
peel-1(A)
X zeel-1(A)
zeel-1 (A)peel-1(A)
C
peel-I activity
Figure 2-1:
bli-3 genotype
+
bli-3(e767)
Present
44
2
Absent
12
41
peel-1 and zeel-1
are genetically separable.
(A) Wild isolate
EG4348 was crossed to Bristol and Hawaii, and lethality was scored among embryos collected from self-fertilizing F1 hermaphrodites and F1 males backcrossed to
Hawaii hermaphrodites. A control cross, using F1 individuals derived from a cross between Bristol and Hawaii, was performed in parallel. (B) Embryo lethality was scored
among embryos collected from three crosses: (i) self-fertilizing zeel-1 (tm3419)/zeel-
1 (+)peel-1 (+) hermaphrodites, (ii) self-fertilizing zeel-1 (tm3419)/zeel-1 (A)peel1(A) hermaphrodites, and (iii) zeel-1 (tm3419)/zeel-1 (A)peel-1 (A) males mated to
zeel-1 (A)peel-i (A)/zeel-1 (A)peel-1 (A) hermaphrodites. The allelic nature of peel-i
on the haplotype carrying zeel-1 (tm3419) is purposefully omitted because the goal of
this experiment was to determine whether the deletion tm3419 disrupts peel-i activity. Expected values were calculated under the hypothesis that tm3419 creates a null
allele of zeel-1 but does not affect peel-1. Among embryos sired by hermaphrodites,
slight decreases from 25% and 100% are expected because the paternal-effect killing
is not fully penetrant when sperm derive from hermaphrodites [115]. (C) Absence
of peel-i activity in EG4348 is genetically linked to bli-3, which is located on the
left-hand tip of chromosome 1, 10 cM from the peel-i interval. EG4348 was crossed
to a strain of the Bristol background carrying bli-3(e767), and F2 chromosomes were
scored for peel-i activity and presence of the bli-3(e767) allele.
31
A
Chromosome I
L5
0 Mb
15 Mb
10 Mb
Mb
2.2 Mb
2.36 Mb
2.34 Mb
peel-1 activity
Present
Recombinants between MY19 and Bristol
-.
Recombinants betweenEG4348 and Bristol
Absent
Present
Bristol
MY19 or EG4348
breakpoint
I Absent
MYl 9 sequencechanges
EG4348 sequencechanges
I iI'l
I I
I
e el-I13-I-I--U-U-I
peel-1H-M
peel-i transgene iZ
tm3 4190l
niDf9 I
B
30
Self-cross
|
I unique to MY19 or EG4348
I shared by wild strains having intact peel-1
nonsense mutationinpeelSp < 10~
60 Backcross
Self-cross Backcross
(n = 193) (n = 220)
25
50
Genot
20
40
zeel-1+/+
24%
n/a
30
zeel-1I-+/A
49%
54.1%
20
zeel-1A/A
1%
0%
unhatched
26%
45.9%
15
10
,,
E 5
W
0
10
peel-i
array
no
peel-1
insertion transgene
0
e
peel-1
array
no
peel-1
insertion transgene
D
MRFDFQNLKFSMIFIFLWNIIVGFLLALVKIFKIYMYLDLPE 4 NVWVNRFARVIALI SIASLWLIFFSPFEIKQGR
frameshift
stop
FSVSSKTILLLVCKGLIGLIFLAQIFAHVLYIDPCQIMLLSLNSALFSLLLNYYESRGAIHPLMELVQTAGLRPTL
CAAVRFNCLNSTAHIDPSVENP
Figure 2-2: peel-1 is located adjacent to zeel-1 and encodes a four-pass
transmembrane protein.
(A) The large black box outlines the genomic region to which peel-1 was mapped
previously [115]. Horizontal bars represent the recombinants used to map the peel-i
mutations in strains MY19 and EG4348. The maximum intervals defined by these
mapping experiments are outlined by white boxes. Adjoining breakpoints are excluded because they contain no sequence changes relative to Bristol. Below the recombinants, tick marks indicate all sequence changes in MY19 or EG4348 located
within the boxed intervals. Horizontal bars represent the peel-i transgene, the zeel1 (tm3419) deletion allele, and the deficiency, niDf9. niDf9 is carried by the Hawaii
strain and by all other wild isolates lacking the activities of both peel-1 and zeel-i
[115].
(B) The Bristol allele of the peel-i transgene shown in (A) was tested for its ability
to restore peel-i activity to animals carrying the peel-i nonsense mutation found in
EG4348. To test for peel-I activity, transgenic animals were crossed to the Hawaii
32
Figure 2-2:
strain, and embryo lethality was scored from self-fertilizing F1 hermaphrodites (selfcross) and F1 males backcrossed to Hawaii hermaphrodites (backcross). Nine independent extra-chromosomal arrays and five independent single-copy genomic insertions
were tested. For each array or insertion, 200 to 650 embryos were scored per self-cross
or backcross. Ten control replicates were performed in parallel, each including 200 to
400 embryos. The global mean of these replicates is shown by the no transgene bars,
and lethality for each individual replicate was less than 1.5%. Error bars indicate
95% binomial confidence intervals, calculated using the Agresti-Coull method [139].
p < 10- 7 E, one-tailed binomial test relative to the mean of the control replicates.
The observed variability among extra-chromosomal arrays was expected because of
germline silencing [66]. The three single-copy insertions showing no peel-i activity
probably represent incomplete insertion events, which are a common outcome of the
MosSCI method [43]. Arrows indicate the insertion used for further analysis in (C).
(C) To confirm that the lethality observed in (B) was limited to zeel-1 (A) embryos, an
additional self-cross and backcross were performed using the insertion marked in (B).
All hatched progeny were genotyped at zeel-1. In both crosses, the genotype frequencies among surviving progeny differed significantly from their Mendelian expectations
(X2 tests, p < 10-9). n/a, not applicable.
(D) The amino acid sequence of PEEL-1. Grey bars indicate predicted transmembrane helices, as predicted by (from top to bottom): TopPred [54], Tmpred [123],
TMHMM [53], SOSUI [112], PHDhtm [137], and HMMTOP [13]. Regions predicted
by at least four algorithms are highlighted in black. The frameshift in MY19 and the
stop codon in EG4348 are indicated in red.
33
breaking apart the peel-i/zeel-i element. We hypothesized, therefore, that MY19
and EG4348 carry secondary, loss-of-function mutations in peel-1, and we reasoned
that by identifying these mutations, we would be able to identify peel-i itself.
To accomplish this goal, we crossed MY19 and EG4348 to a strain of the Bristol
background and generated recombinant chromosomes across the peel-I interval. Using
these recombinants, we mapped the causative mutations in MY19 and EG4348 to
regions of less then 10 kb (Figure 2-2 A). We then sequenced these regions to identify
all sequence changes relative to Bristol. After excluding those sequence changes shared
by one or more wild strains having intact peel-i activity (Figure 2-2 A, Figure 2-3), we
defined six candidate mutations in MY19 and a single candidate mutation in EG4348.
The candidate mutations in MY19 and EG4348 reside in the intergenic interval
immediately downstream of zeel-i (Figure 2-2 A). We searched for genes in this
interval using targeted RT-PCR on Bristol animals, and we discovered a previously
unannotated transcript. This transcript encodes a 174-amino acid protein (Figure
2-2 A,D), and three observations confirm this gene to be peel-1. First, the single
candidate mutation in EG4348 and one of the candidate mutations in MY19 produce
nonsense mutations in this transcript, consistent with the phenotype of these strains
(frameshift in MY19; glycine to stop codon in EG4348) (Figure 2-2 A,D; Figure 2-3).
Second, this gene is absent in the Hawaii strain and in all other wild isolates lacking
the activities of both peel-i and zeel-1 (Figure 2-2 A). Third, when we expressed the
Bristol allele of this gene transgenically in a strain carrying the nonsense mutation
found in EG4348, peel-i activity was restored (Figure 2-2 B-C).
PEEL-1 is a hydrophobic protein containing four predicted transmembrane helices
(Figure 2-2 D). Neither the peptide nor the nucleotide sequence of peel-i has any
detectable sequence similarity to any other gene in C. elegans or in the GenBank
sequence database. Although peel-i and zeel-i are located adjacent to one another in
the genome and oriented in the same direction, we were unable to recover transcripts
carrying both genes (data not shown), demonstrating that peel-i and zeel-i are not
isoforms of a single transcript or cistrons in an operon. We conclude that the peelI /zeel-i element is composed of a 19 kb insertion carrying two distinct genes: peel-1,
34
which kills embryos via paternal-effect; and zeel-1, which acts zygotically to rescue
this lethality.
Henceforth we refer to the Bristol alleles of peel-i and zeel-1 as peel-i(+) and
zeel-1(+) and the Hawaii alleles as peel-i(A) and zeel-i(A). We use the term peel-1affected embryos to refer to zeel-i (A) embryos fathered by a peel-i(+) animal.
peel-1 mRNA is expressed in spermatocytes, and the protein localizes to
fibrous body-membranous organelles
As expected for a paternal-effect gene, peel-1 is expressed exclusively in sperm. In
both males and hermaphrodites, a GFP reporter driven by the peel-i promoter was
expressed strongly in spermatocytes but not in any other tissue (Figure 2-5 A). In
fem-i (ts) mutants, which lack sperm [22], peel-i expression via quantitative RT-PCR
was undetectable in hermaphrodites and over 100-fold reduced in males (Figure 2-5
C). Residual expression in males probably reflects incomplete penetrance of the femi(ts) allele, because fem-i(ts) males occasionally produce a small number of sperm
[22], and another sperm-specific gene, spe-9 [122], also showed residual expression in
males (Figure 2-5 C).
The sperm-specific expression of peel-i suggested that the paternal-effect killing
occurs through delivery to the embryo of either sperm-supplied peel-i transcripts or
sperm-supplied PEEL-1 protein. To distinguish between the two, we searched for peel1 transcripts in mature sperm via single-molecule fluorescence in situ hybridization
(FISH). peel-i transcripts were observed in spermatocytes but not in mature sperm
(Figure 2-4), thus excluding such transcripts as the likely mediators of peel-i toxicity.
Next, we searched for PEEL-1 protein both by tagging PEEL-1 with GFP and
by staining sperm with an antibody raised against the C-terminal 15 amino acids of
PEEL-1. Both experiments demonstrate that PEEL-1 protein is packaged into sperm,
and this packaging is mediated by localization of PEEL-1 to sperm-specific vesicles
called fibrous body-membranous organelles (FB-MOs) (Figure 2-6 A). PEEL-i::GFP
was visible in mature sperm (Figure 2-6 E-F), and at each stage of spermatogenesis,
its localization matched the pattern expected for a protein located in the membranes
35
Functional
4
Wild isolate
%S
N2 (Bristol)
T
A
T
A
G
G
A
G
T
T
go'
EG4348
AB1
CB3196
CB34852
CB4932
DR1344
DR1349
ED3014
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%S
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%S
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peel-I coding
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Figure 2-3: Some MY19 and EG4348 sequence changes are shared with wild
isolates having intact peel-1. All of the sequence changes in MY19 and EG4348
located within the boxed intervals shown in Figure 2-2 A were genotyped in a panel
of 38 wild strains shown previously to have intact peel-1 activity [115]. The position
of each polymorphism (WormBase release May 2008 WS190/ce6) and the primers
used to amplify and sequence it are listed diagonally above each column. Alleles
unique to MY19 are shown in pink, alleles unique to EG4348 are shown in cyan, and
alleles shared by at least one additional wild strain are shown in grey. Polymorphisms
affecting the amino acid sequence of peel-i are indicated in the bottom row. n/d, not
determined; (-), single base-pair deletion.
36
"
of FB-MOs: in early spermatocytes, PEEL-1::GFP localized to cytoplasmic puncta
(Figure 2-6 B), but after the pachytene stage, these puncta dissociated into a meshlike web (Figure 2-6 B-D); after the completion of spermatogenesis, PEEL-i::GFP recondensed into puncta located at the spermatid cortex (Figure 2-6 E); and after sperm
activation, these puncta localized opposite the newly formed pseudopod (Figure 2-6
F). This localization pattern was replicated by the anti-PEEL-i antibody (Figure 2-6
G), and staining with this antibody overlapped perfectly with a marker of FB-MOs
(Figure 2-6 H-I).
We also discovered that the leader peptide of PEEL-I can act as a sperm-localization
signal. Our Ppeel-1::GFPreporter, which expressed untagged GFP, showed diffuse
localization in spermatocytes and was excluded from sperm (Figure 2-5 B). This exclusion was not surprising because GFP is a heterologous protein, and trafficking
of cellular components into sperm is tightly regulated [16]. Nevertheless, when we
tagged GFP with the N-terminal 12 amino acids of PEEL-1 (MRFDFQNLKFSM),
its localization changed dramatically. The tagged version of GFP localized to a reticulated structure within spermatocytes, and this structure was trafficked into sperm
(Figure 2-5 B). To our knowledge, this result represents the first identification of a
sperm localization signal in C. elegans.
peel-1-affected embryos show late-stage defects in muscle and epidermal
tissue
Unlike other known paternal-effect genes [21, 42], sperm-supplied PEEL-i does not
cause defects until late in development. In peel-i-affected embryos, early embryogenesis, gastrulation, epidermal enclosure, and early elongation occur normally (Figure
2-7 C). Then, at the two-fold stage of elongation, when all major tissues have begun differentiating and nearly all embryonic cell divisions have already occurred, the
majority of peel-1-affected embryos arrest elongation and fail to begin rolling within
their eggshells. Shortly thereafter, the bulk of the embryo compresses inward, towards the mid-embryo bend, and the head and tail become flaccid and thin (Figure
2-7 D). Approximately two hours later, cytoplasm begins leaking from the external
37
...-s perm
..-s perm atocyte
Figure 2-4: peel-1 mRNA is not present in sperm. peel-i mRNAs were visualized in a wild-type, L4 hermaphrodite using single-molecule fluorescence in situ
hybridization. peel-1 mRNAs are shown in red, and nuclei are stained with DAPI
(cyan). Sperm and spermatocyte nuclei are labeled.
38
Seconidary
spermia to cy te
C
u
10
r/s
I
0)
C
Bud dini gpri~td
s peir miatid s
*
..
1
a/)
L-
0.1
a)
0.01
[peel-I
0 spe-9
25'C
15'C
him-5
~
150C
25'C
him-5; fem-1(ts)
Genotype and temperature
Figure 2-5: peel-1 is expressed exclusively in sperm and carries an Nterminal sperm localization signal.
39
Figure 2-5:
(A) Adult male expressing Ppeel-1::GFP.The tube-like gonad begins at the asterisk, extends toward the head, folds over on itself, and then extends toward the tail.
The arrow and arrowhead indicate spermatocytes and sperm, respectively. Fluorescence outside the gonad is auto-fluorescence. Nomarski and fluorescence channels are
overlaid.
(B) Secondary spermatocytes, budding spermatids, and mature spermatids expressing
either untagged GFP or GFP tagged with the N-terminal 12 amino acids of PEEL-1
(PEEL - 112a.a. ::GFP). Arrows indicate residual bodies. Nomarski and fluorescence
channels are overlaid.
(C) Relative expression levels of peel-1 and spe-9 in him-5(e1490) and him-5(ei490)
fem-1(hc17ts) adult males at the permissive (15C) and restrictive (25C) temperatures. him-5(ei490) was included to aid in collection of males. Expression levels
were calculated relative to the him-5(ei490) fem-i(hci7ts) 15C sample. Runs were
performed in triplicate and standard deviations are shown. *, p < 10-5, one-tailed
Students t-test on the normalized expression levels. n/s, p > 0.05.
epidermis, and the lumen of the excretory cell distends to form large vacuoles (Figure
2-7 D).
The defects observed in peel-1-affected embryos indicate severe malfunction of
muscle and epidermal tissue. The phenotype of paralysis and two-fold arrest is characteristic of a complete absence of the function of body-wall muscle [90, 145]. The
shape changes observed after the two-fold arrest indicate detachment of body-wall
muscle fibers from the overlying epidermis [14]. Epidermal leakage and distention of
the excretory cell, the only epidermal cell located in the interior of the animal, indicate further deterioration of both external and internal epidermis. These four defects
paralysis and two-fold arrest, muscle-epidermal detachment, epidermal leakage, and
excretory cell distention are not known to occur as side-effects of one another [145, 71],
suggesting that sperm-supplied PEEL-1 may affect each tissue independently.
Paralysis and two-fold arrest have only two known causes: defective sarcomere assembly and lack of muscle contraction [90, 145]. To distinguish between the two, we
examined (i) the localization of perlecan, a basement membrane protein required for
sarcomere recruitment [145]; and (ii) the structure of actin and myosin myofilaments,
which assemble downstream of all other sarcomere proteins [90]. In peel-1-affected
embryos, perlecan localized normally (Figure 2-7 I-J). Likewise, actin and myosin
40
A
syncytial
gonad
'
'''
/
0_
0
*
0
fibrous bodymembranous organelle
IE
Figure 2-6: PEEL-1 localizes to fibrous body-membranous organelles. (A)
Diagram of spermatogenesis and fibrous body-membranous organelle (FB-MO) development, adapted with permission from [77]. FB-MOs develop in pachytene spermatocytes as membrane-bound organelles having a head region separated by a collar-like
constriction from a set of membrane folds (lower panel; red). As spermatogenesis
proceeds, the membrane folds grow and extend into arm-like protrusions, enveloping
bundles of polymerized Major Sperm Protein, referred to as fibrous bodies (hatched
region). Coincident with budding of spermatids from the residual body, the membrane
folds of FB-MOs retract, and the fibrous bodies depolymerize into the cytoplasm. The
FB-free MOs then move to a position just inside the plasma membrane, and upon
sperm activation, they fuse with the plasma membrane opposite the pseudopod.
(B-F) Nomarski and fluorescence images of spermatocytes and sperm expressing
PEEL-1::GFP. Panels in (B) show the proximal arm of a male gonad, oriented with
pachytene spermatocytes towards the left (bracketed region). Arrow and arrowhead
indicate primary and secondary spermatocytes, respectively. Panels in (C-F) show
higher resolution images of the following stages: secondary spermatocyte (C); budding spermatids (D); unactivated spermatid (E); and activated spermatozoan (F).
(G-K) Spermatids were dissected from peel-1(+) (G-I) or peel-1(A) (J-K) males and
stained with anti-PEEL-i (green) and the FB-MO marker, 1CB4 (red) [96]. Nuclei
are stained with DAPI (blue).
41
filaments assembled correctly, except for slight abnormalities in regions of muscle
detachment (Figure 2-7 K-N). We conclude that in peel-i -affected embryos, the phenotype of paralysis and two-fold arrest results from a defect in muscle contraction,
not sarcomere assembly.
Next, to determine the cause of muscle-epidermal detachment, we examined transepidermal attachments, the specialized structures that span the epidermal syncytium
and anchor it to underlying muscle
[711.
A weakening of these structures is known to
cause muscle-epidermal detachment [26], and consistent with this causality, in peel1-affected embryos these structures were highly disorganized. As visualized by VAB10A and intermediate filaments, trans-epidermal attachments did not organize into
evenly spaced, circumferentially oriented bands. Instead, these bands were clumpy,
disordered, and non-uniform in width (Figure 2-7 O-R). This disorganization occurred
even in areas where muscles remained attached, suggesting it to be the cause of muscle
detachment, rather than an effect of it. In addition, in areas highest stress, such as
the mid-embryo bend, staining in post-arrest embryos (but not pre-arrest embryos)
was often absent altogether (Figure 2-7 P). This absence implies that trans-epidermal
attachments in peel-1-affected embryos are so weak that in areas of highest stress,
they rupture entirely.
peel-1 toxicity is dose-dependent
Although the majority of peel-1-affected embryos display the aforementioned defects
in muscle and epidermal tissue, the severity of these defects is variable and depends
on the sex [115] and age of the sperm parent.
Male-sired embryos always arrest
paralyzed at the two-fold stage, always exhibit epidermal leakage, and never hatch
(n > 2,000). Some hermaphrodite-sired embryos, on the other hand, elongate past
the two-fold stage (Figure 2-7 E) or do not exhibit epidermal leakage.
Occasion-
ally, hermaphrodite-sired embryos even hatch, and the hatched progeny range from
severely deformed larvae that die soon after hatching (Figure 2-7 F) to morphologically normal larvae that develop into viable, fertile adults. In addition, among
hermaphrodite-sired embryos, the proportion of embryos arresting at the two-fold
42
'D
KW
1O
P
R
CQ
Figure 2-7: peel-1-affected embryos exhibit late-occurring defects in muscle and epidermal tissue. (A and B) Wild-type embryo at the 1.5-fold stage
(A) and just before hatching (B). (C and D) peel-I-affected, male-sired embryo at
the 1.5-fold stage (C) and approximately four hours after the two-fold arrest (D).
Relative to its shape at the two-fold stage, the embryo in (D) has shortened longitudinally and thickened circumferentially. Thinning of the tail, distention of the
excretory cell (arrow), and epidermal leakage (arrowheads) are visible. (E and F)
peel-I-affected, hermaphrodite-sired embryos displaying less severe phenotypes than
the embryo shown in (D). In (E), the embryo has elongated past the two-fold stage,
but muscle detachment is visible (arrow). In (F), the embryo has hatched but is
severely deformed.
(G-H) peel-1-affected, male-sired embryos expressing zeel-1 in
only muscle (G) or only in epidermis (H). The embryo in (G) has elongated past
the two-fold stage, but epidermal leakage is visible (arrow). The embryo in (H) has
arrested paralyzed at the two-fold stage but has survived to hatching. (I-N) Perlecan,
myosin heavy chain A, and F-actin were visualized in wild-type and peel-1 -affected,
male-sired embryos. In peel-1-affected embryos, muscle detachment is evident at the
mid-embryo bend, where muscle fibers (arrows) are displaced inward relative to their
proper locations (dashed lines). (O-R) VAB-10A and intermediate filaments were visualized in wild-type and peel-I-affected, male-sired embryos. In peel-1-affected embryos, VAB-10A and intermediate filaments are recruited properly to the four muscle
quadrants, but they do not organize into evenly spaced, circumferentially oriented
bands. In (P), VAB-10A staining is absent in one dorsal quadrant at the mid-embryo
bend (arrow). Images in (0-P) are dorsal views.
43
stage and the proportion failing to hatch decreased dramatically with parental age
(Figure 2-9 A-B, Figure 2-8). Among male-sired embryos, phenotypic severity was
unaffected by parental age (Figure 2-9 C).
One explanation for the decreased phenotypic severity of hermaphrodite- versus
male-sired embryos is PEEL-1 dosage. Male sperm are up to five-fold larger than
hermaphrodite sperm [73], and as such, they may deliver more PEEL-1 protein to
the embryo. In support of this hypothesis, we were able to alter the phenotype of peel1-affected embryos, independent of sperm origin, by varying peel-i dosage. Among
hermaphrodite-sired embryos, doubling peel-1 copy number resulted in earlier onset
of epidermal leakage (Figure 2-9 D). Among male-sired embryos, expression of peel-1
exclusively from extra-chromosomal arrays, which are subject to germline silencing
[66], had the opposite effect. For three of the peel-i arrays shown in Figure 1B, 3-10%
of male-sired embryos elongated past the two-fold stage and hatched into deformed
larvae (n > 150 embryos per array). When the same peel-i transgene was expressed
from a single-copy genomic insertion, which is not silenced, no hatching was observed
(Figure IC).
Given the relationship between PEEL-1 dosage, sperm size, and phenotypic severity, we suspected that the age-related decrease in phenotypic severity among hermaphroditesired embryos might reflect underlying size differences and size-based competition
among hermaphrodite sperm. Hermaphrodite sperm vary approximately two-fold in
size
[731
and are produced in a single bout of spermatogenesis at the onset of adult-
hood. Larger sperm in C. elegans experience a competitive advantage because they
are able to crawl faster to reach the oocyte [73, 121]. One explanation for the age effect, therefore, is that larger-than-average sperm monopolize fertilization events early
in life, leaving smaller, less toxic sperm to dominate fertilizations later on. In support
of this hypothesis, we were able to reduce the age effect among hermaphrodite-sired
embryos by delaying the hermaphrodites use of self-sperm via partial mating to a
male (Figure 2-9 B). This result demonstrates that the age effect arises from a correlation between the competitive ability of each sperm and its toxicity to the embryo.
Given the known biology of C. elegans sperm, the most parsimonious explanation for
44
this correlation is that larger hermaphrodite sperm both are more competitive and
carry more PEEL-1 protein. This correlation might also arise from PEEL-1 having a
direct effect on the competitive ability of each sperm, although we have no evidence
for such an effect.
To our knowledge, the above results represent the first evidence of competition
among hermaphrodite sperm in vivo, as well as the first evidence of naturally occurring differences in sperm size affecting embryonic development. Insofar as PEEL-1
levels scale with sperm size, the wide phenotypic variability among hermaphroditesired embryos implies that for low levels of PEEL-1, phenotypic severity is acutely
sensitive to PEEL-1 dosage. By the same logic, however, the phenotypic homogeneity
among male-sired embryos implies that above a certain threshold level of PEEL-1,
phenotypic severity ceases to increase. In support of this threshold effect, doubling
or tripling peel-i copy number among male-sired embryos did not produce a more
severe phenotype, at least as measured by the onset of epidermal leakage (Figure 2-9
D).
zeel-1 is expressed transiently in the embryo, and tissue-specific expression
of zeel-1 produces tissue-specific rescue
Consistent with its function as an antidote to sperm-supplied PEEL-1, zeel-1 is expressed in the embryo. Yet its expression is transient. By single-molecule FISH,
zeel-1 expression begins at the eight-cell stage, peaks at approximately the 150-cell
stage, and then turns off (Figure 2-10 A). Transient expression was also observed for a
GFP-tagged version of ZEEL-1, whose levels peaked during mid-embryogenesis (Figure 2-11), but whose expression was not observed in late-stage embryos, nor larvae
or adults (data not shown).
Within embryos, ZEEL-1::GFP was expressed in all or almost all cell types (Figure
2-10 B-C, Figure 2-11). The protein localized most strongly to cell membranes (Figure
2-10 B-C), consistent with ZEEL-1 having an N-terminal transmembrane domain. In
some tissues, such as the developing pharynx and intestine, ZEEL-i::GFP appeared
more concentrated at the apical face (Figure 2-10 C).
45
100
41 52
40 71 89
85
80
75
60
40
20
0i Broodi1
100
-
E
33 63
ry/a
Brood 4
4
41
16 4
-77
73 1
80
60
40'
18'
1
20
0
Brood 6
.
-
Brod7
I
0- 1- 1.5- 2- 2.5- 31d 1.5d 2d 2.5d 3d 5d
9
Br BrBro
0- 1-1.5- 2- 2.5- 31d 1.5d 2d 2.5d 3d 5d
0- 1-1.5- 2- 2.5- 31d 1.5d 2d 2.5d 3d 5d
Hermaphrodite age (days post-L4)
Figure 2-8: Age-related decrease in the lethality of peel-1 -affected,
hermaphrodite-sired embryos in ten randomly selected broods. The results for ten of the 91 broods used in the unmated experiment in Figure 2-9B are
shown. Broods were selected using a random number generator. As described in
Figure 2-9B, each hermaphrodite was followed from the onset of adulthood, and all
embryos laid during the first five days of adulthood were collected. n/a, no embryos
laid during this time period.
46
A
n/s
B
100, 3538
100
5899
*
7828
80
80
n.
Csoq
60
0
40
o40
20
E 20
0
1
6091
01
1d
2d 2.5d 3d
Hermaphrodite age
(days post-L4)
0-1d 1-1.5d 1.5-2d 2-2.5d 2.5-3d 3-5d
3-5d
unmated
partially mated
Mating status and hermaphrodite age
(days post-L4)
C
n/s
D
Aget
!f 3
% Embryo
lethality (total)
>7 or
no leakage
7h
n = 80
85
1d
I
100% (n>1200)
5h
3d 3d
100% (n>1200)
5d
100% (n>500)
*
t days post-L4
* sperm aged for 3 days
within the reproductive
tract of the hermaphrodite
88
0CD
6h
1d
45
60
I
I
U 4h
15
I
I
3h
S6 2h
ih
+/+
+/+/+
Male
Sex and peel-I genotype of sperm parent
+/A
+/+
+/A
Hermaphrodite
Figure 2-9: The phenotypic effects of sperm-supplied PEEL-1 are dosedependent.
(A) The proportion of embryos arresting at the two-fold stage was calculated among
peel-i-affected embryos sired by one- to three-day-old hermaphrodites. Within each
age class, embryos derive from a total of approximately 50 to 150 hermaphrodites.
All pair-wise combinations of age classes were compared using X2 tests. For all pairs,
p < 10-5.
(B) Embryo lethality was scored among peel-1-affected embryos sired by unmated,
one- to five-day-old hermaphrodites and by partially mated, three- to five-day-old
hermaphrodites. In the unmated experiment, 91 hermaphrodites were followed from
the onset of adulthood, and all embryos laid during the first five days of adulthood
were scored. The results for ten randomly selected broods are shown in Figure 2-8. To
generate embryos sired by partially mated animals, 130 hermaphrodites were briefly
47
Figure 2-9:
mated to males following the L4 molt, and embryos were collected during days three
to five from the 35 hermaphrodites that produced a mixture of self- and cross-progeny.
Self- and cross-progeny were distinguished by the use of an integrated GFP marker
carried by the male, and cross-progeny were excluded from analysis. X2 tests were
used to compare the unmated and partially mated 3-5d age classes, as well as all
pair-wise combinations of age classes within the unmated experiment. n/s, p > 0.05.
* and all unlabeled pairs within the unmated experiment, p < 10-5.
(C) Embryo lethality was scored among peel-i -affected embryos derived from crosses
between i) one-day-old males and hermaphrodites; ii) three-day-old males and
hermaphrodites; and iii) five-day-old hermaphrodites that had been removed from
males on day two in order to allow male sperm to age for three days within the reproductive tract of the hermaphrodite. In each cross, embryos derive from a total of
12 to 16 hermaphrodites.
(D) Spindle plots showing the onset of epidermal leakage in peel-i-affected embryos
sired by hermaphrodites carrying one or two copies of peel-i (+), or by males carrying
one, two, or three copies of peel-i(+). A third copy of peel-i was added using the
single-copy insertion of the peel-1 transgene marked in Figure 1B. The width of each
bar reflects the proportion of embryos initiating leakage in each time interval. All
pair-wise combinations of spindle plots were compared using Mann-Whitney U tests
on the raw distributions of leakage times. n/s, p > 0.05. For all other pairs, p < 10-.
Ubiquitous zeel-I expression is consistent with zeel-Is ability to rescue the seemingly independent muscle and epidermal defects observed in peel-i-affected embryos.
To test the tissue-autonomy of zeel-i rescue, we expressed zeel-i only in muscle
and only in epidermis.
We used the promoters of hlh-i and lin-26, respectively,
[69, 74], which initiate expression at the 80- to 100-cell stage [72]. Consistent with
sperm-supplied PEEL-1 affecting muscle and epidermis independently, tissue-specific
expression of zeel-I produced tissue-specific rescue. In male-sired embryos, expression
of zeel-I only in muscle rescued the muscle defect of paralysis and two-fold arrest,
but it did not rescue epidermal leakage (n = 120 embryos; Figure 2-7G). (Muscle
detachment and excretory cell distention could not be assayed because muscle movement, combined with epidermal leakage, ripped embryos apart entirely.) Conversely,
expression of zeel-i only in the epidermis rescued the epidermal defects, but it did not
rescue paralysis and two-fold arrest (n = 69 embryos; Figure 2-7H). In hermaphroditesired embryos, both constructs fully rescued a subset of embryos, and rescue activity
increased with hermaphrodite age (Figure 2-12). This result is consistent with the
48
age effect among hermaphrodite-sired embryos and the fact that hermaphrodite-sired
embryos do not always exhibit defects in both muscle and epidermal tissue.
The transmembrane domain of zeel-1 is evolutionarily novel and partially
sufficient for antidote activity
The structure of zeel-1 -
a C-terminal region (-700 amino acids) homologous to the
highly conserved gene, zyg-11, and an N-terminus (~200 amino acids) predicted to
form a six-pass transmembrane domain is highly unusual, and phylogenetic analysis
indicates that this structure arose during a recent expansion of the zyg-11 family.
Most metazoan genomes contain one to two zyg-11 orthologs, but in C. elegans, C.
briggsae and C. remanei, the zyg-1I family has expanded such that these species carry
19 to 37 zyg-Il-like genes each (Figure 2-14). Most of these additional family members
probably arose after the split with out-group C. japonica, because the genome of C.
japonica contains only three zyg-11-like genes (including Cja-zyg-11 itself).
Aside from zeel-I, only two other members of the zyg- 11 family paralogs Y71A 12B. 17
and Y55F3C.9
contain predicted transmembrane domains (Figure 2-13 A, 2-12).
The transmembrane domains of these three genes are homologous to one another,
but these domains show no detectable sequence similarity to any other gene in C.
elegans or in the GenBank sequence database.
This pattern, combined with the
closest-paralog relationship between the soluble domains of zeel-1, Y71A12B.17, and
Y55F3C.9 (Figure 2-13 A, 2-12) implies that their shared transmembrane domain
is evolutionarily novel and originated after the split between C. elegans and the C.
briggsae/ C. remanei lineage.
Analysis of gene order and sequence data suggests that zeel-1 arose through duplication of the Y71A12B.17 locus. zeel-1 and Y71A12B.17 are one anothers closest
paralogs (Figure 2-13 A, 2-12), and the two genes are 55% identical at the amino
acid level.
Y71A12B.17 is located 12 Mb from zeel-1 in a tandem array of three
additional zyg-11 family members, none of which contain the N-terminal transmembrane domain. Assuming that these four genes originated in their current genomic
location via repeated tandem duplication, then two scenarios for the origin of zeel-1
49
A
-5 200
40
A1
z
Ez
W100
E
It
0
0
10
20
number of cells
jj 50
100 150200 end of comma
number of cells gastrulation
(~250 cells)
Developmental stage
2-fold >3-fold
Figure 2-10: zeel-1 is transiently expressed during embryogenesis and localizes to cell membranes. (A) zeel-1 mRNAs were quantified in wild-type embryos
via single-molecule fluorescence in situ hybridization [105]. Embryos were staged by
counting nuclei manually (1- to 40-cell embryos); counting nuclei using image analysis software (41- to 200-cell embryos); or classifying embryos as end of gastrulation
(-250-cells), comma, 2-fold, or > 3-fold. Inset shows a magnification of the boxed
area. Each circle represents an independent embryo. n = 130. (B-C) Embryos
expressing ZEEL-1::GFP.Panel in (B) shows a dorsal view during intercalation of
epidermal cells. Arrow indicates an epidermal cell membrane. Panel in (C) shows a
lateral cross section of a 1.5-fold embryo. The apical face of the pharynx (arrow) and
the intestine (arrowhead) are indicated.
50
V
hours post 4-cell stage:
number of cells:
0h
4
V
1h
-25
V
2h
-50
V
3h
-150
V
4h
-300
5h
-400
I'lt
6h
7 h
8h
9h
14 h (hatch)
-550 - - - - - - - - - - - - - - - - - - - - - - - - -
Figure 2-11: ZEEL-1::GFP is expressed transiently during embryogenesis.
Time series images of a single embryo expressing ZEEL-1::GFP under the zeel-1 promoter. Timeline indicates embryo age in hours post-4-cell stage.
and Y71A12B.17 are possible. First, Y71A12B.17 may have originated via duplication of another gene in the tandem array, gained its transmembrane domain during
or after duplication, and later been duplicated again to produce zeel-1. Alternatively,
the tandem array may have arisen through partial duplication Y71A12B.17. The
second scenario is less parsimonious than the first, because it requires secondary loss
of the transmembrane domain during creation of the tandem array. However, the
second scenario still implies that the Y71A12B.17 locus predates zeel-1, because if
the opposite were true, then zeel-1 would form an out-group to the tandem array,
and it does not (Figure 2-13 A).
Given the chimeric structure of ZEEL-1, we tested whether either domain alone
could rescue the lethality of peel-i-affected embryos. The soluble ZYG-11-like domain, ZEEL - 1 SOL, provided no rescue (Figure 2-13B). The transmembrane domain,
ZEEL - 1 TM, provided full rescue to hermaphrodite-sired embryos, but only partial
rescue to male-sired embryos (Figure 2-13B-C). In contrast, the positive control transgene, full-length ZEEL-1 tagged with GFP, provided full rescue to both male- and
hermaphrodite-sired embryos (Figure 2-13B-C). We conclude that the transmembrane
domain of ZEEL-1 is required for antidote activity, and that this domain alone is able
to neutralize the low doses of PEEL-1 delivered by hermaphrodite sperm but not the
higher doses delivered by male sperm.
The partial rescue activity of ZEEL -
'TM
demonstrates that ZEEL -
1
SOL
does contribute to the antidote activity of full-length ZEEL-1. To examine this contribution more carefully, we tested whether ZEEL 51
1
SOL
could rescue the lethality
inherited transgene
did not inherit transgene
Phlh-1::zeel-I
Plin-26::zeel-I
n=
n=
100.
,
*
*
100
.
Q7
*
163
184
80
80
837
60
60
40
40
20
20
.5
0
745
E
w
171
793
9
724
621
0
1-1.5d
1.5-2d
1-1.5d
2-2.5d
1.5-2d
2-2.5d
Hermaphrodite age (days post-L4)
Figure 2-12: Tissue-specific expression of zeel-1 is partially sufficient for
rescue of peel-1 -affected, hermaphrodite-sired embryos. Embryo viability
was calculated among peel-1 -affected, hermaphrodite-sired embryos inheriting either
Plin-26::zeel-1 or Phlh-1::zeel-1. Embryos are grouped according to the age of the
parent hermaphrodite. White bars indicate sibling controls that did not inherit the
transgene. *, p < 10-5, x 2 tests.
52
of peel-1-affected embryos when fused to the transmembrane domain of zeel-is closest relative, Y71A12B.17. Like ZEEL TM::ZEEL -
1
SOL,
1
SOL,
the chimeric transgene, Y71A12B.17
provided no rescue (Figure 2-13B). Assuming that this trans-
gene was stably expressed, this result demonstrates that ZEEL -
1
SOL cannot con-
fer antidote activity to a related transmembrane domain. Additionally, this result
demonstrates that the transmembrane domains of zeel-1 and Y71A12B.17 have diverged functionally since their common ancestor. The molecular basis of this divergence remains unclear, however, because the transmembrane domains of zeel-1
and Y71A12B.17 are only 35% identical at the amino acid level, with substitutions
distributed throughout their length (Figure 2-14).
Heat-shock expression of peel-1 kills adult animals, and heat-shock expression of zeel-1 rescues this lethality
To determine whether PEEL-1 can function as a toxin outside of embryos, we expressed peel-i ectopically in larvae and adults. peel-i was expressed using each of
two heat-shock promoters, Phsp-16.2 and Phsp-16.41
[64].
For each promoter con-
struct, we generated both single-copy genomic insertions and extra-chromosomal arrays, which typically contain tens to hundreds of copies of a transgene
[85].
Both
types of animals grew normally under standard laboratory conditions, but a one-hour
heat shock at 34C was lethal to all: array-carrying adults died within two hours after the start of heat-shock, and insertion-carrying animals within 4.5 hours (Figure
2-15 A). Faster killing of array-carrying animals is consistent with their higher peel-i
dosage, and similar results were observed for heat-shocked larvae (data not shown). In
addition, aside from the gross phenotype of death, the heat-shocked animals showed
defects in most, if not all, tissues. Beginning approximately 30 to 45 minutes before death, the body-wall and male-tail muscles hyper-contracted; vacuoles formed
in many tissues (Figure 2-16 K); the lumen of the excretory cell distended (Figure
2-16 L); the gonad appeared to disintegrate (Figure 2-16 M); and in hermaphrodites,
the gonad and intestine occasionally exploded through the vulva. We conclude that
PEEL-1 is a nearly universal toxin, affecting many developmental stages and cell
53
A
Self-cross
B
99
1
Y55F3C.9
26
100
_100j
100
1 2
no transgene
Y7IA12B.17
Y71AI2B.15
-Y71 A12B3.18*
Y71A128.12
F47G4.2
K05C4.9
1
v.s. 'no
ZEEL-10L
Y71A12B.17
-F44E.2
ZE
transgene':
34
N p < 10^
E-p>0.05
0P>00
1V-
ZEL1sOL
0 5 10 15 20 25
0 10 20 30 40 50
Embryo lethality (%)
% Hatched (total embryos)
F1C2.12
F
5
-:
F58D2.1
C48D1.1 a
C50F3.1
100
0.4
4
] n/s
ZEEL-1T
04D03.4
H24K24.2
29
ZEEL-1::GFP
F53G2.1
23
Backcross
2W06A11.2
Y9C9A13
Sperm parent
ZEEL-TM
ZEEL-1::GFP
Hermaphrodite
99.1% (939)
97.6% (705)
p > 0.05
Male
36.4% ( 3 71)t
95.4% (151)
p < 10-5
tMost hatched
tZYG-11
progeny were severely deformed
Figure 2-13: The transmembrane domain of zeel-1 is evolutionarily novel
and partially sufficient for function.
(A) Maximum likelihood phylogeny of the protein sequences of all zyg-11 homologs
in C. elegans. Genes containing predicted transmembrane domains are highlighted in
black. The transmembrane domains of these genes were excluded prior to analysis.
Genes located in the tandem array are highlighted by a shaded grey rectangle. Scale
bar indicates amino acid substitutions per site. Values on branches indicate percent
bootstrap support. The asterisk indicates that the reference sequence of Y71A 12B. 18
contains a single frame-shift, corrected prior to analysis.
(B) Four zeel-1-derived transgenes were introduced into a strain carrying the zeel-1
deficiency, niDf9, and tested for their ability to rescue peel-1-affected embryos. To
test for rescue, transgenic animals were crossed to the Bristol strain, and lethality was
scored among embryos derived from self-fertilizing F1 hermaphrodites (self-cross) and
F1 males backcrossed to hermaphrodites of the original transgenic line (backcross).
For each type of transgene, four to thirteen independent extra-chromosomal arrays
were tested. For each array, 100 to 600 embryos were scored per self-cross or backcross.
Ten control replicates were performed in parallel, each including 100 to 400 embryos
(no transgene bars). Among arrays or control replicates, lethality scores were averaged
to obtain global means and standard deviations. Each transgene was tested for a
reduction in lethality compared to the control replicates (Students t-tests, p-values
indicated by shading). Additionally, the two rescuing transgenes (ZEEL-1TM and
ZEEL-1::GFP)were tested for significant differences relative to one another (Students
t-tests; *, p < 0.005; n/s, p > 0.05). For the rescuing transgenes, lethality was not
reduced to zero because extra-chromosomal arrays are not transmitted to all progeny.
(C) Hatch rates were compared between peel-I-affected embryos that we confirmed to
have inherited either ZEEL-TM or ZEEL-1::GFP (X2 tests, p-values shown). Separate comparisons were performed for male- and hermaphrodite-sired embryos. Unless
otherwise specified, all hatched progeny appeared morphologically normal. Inheritance of the transgenes was determined by expression of the co-injection marker,
Pmyo-2::RFP. Sibling embryos not inheriting the transgene were used as internal
negative controls. The hatch rates of these controls were 2% (n = 601 - 653) among
hermaphrodite-sired embryos and 0% (n = 341 - 933) among male-sired embryos.
54
B
ZEEL-ITM
Y71AI2B. 1 7 TM
MPDLEEA-----NDWSCDFFVLTLIYITSMVFAGVCFSLTNTVKHCVEQHACTNDGKYLVIALVFTVIGLVAFFTVYKLYNWNA
ZEEL-ITM
Y7IA12B. 1 7 TM
KKVYLPLLTFHRNCIVAFSLMLLLYQFIESDV-VLCLYSASWGFLIACYIFSTRVQIASFVMTEDEVMCM
ZEEL-1TM
Y7IA12B. 1 7 TM
IQGAIYGKIIHEISIRGFNLGWFIVVHALVFFQLNVNALKKNERKEG
MSDFDSEERPTFDGWSCRLGFMLLTF-NLLVITGIATGI SNMIRICVDPPECXTGEVYVI IALVI IFLGFI GFLIGYNFYNW-T
--- LFIFFLTLMCS
KKMYLPLLTLNRASIVTFGFLLLIYQAIDSNVFLLYLYSISWGLIAACYFNSLCAQLVSSIIPEERLIILNGYLCIFF----SG
identical
AAGFGLDRFTVKLEYGGFKLGSFILLGPLVSYLIT YIVSKNERKEG
-sit
:
1 ::
::*
I!::: :I:
conserved
Semi-COnSerVed
Figure 2-14: The zyg-11 family has expanded in C. elegans, C. briggsae,
and C. remanei.
(A) PhyML [49] was used to construct a maximum likelihood phylogeny of the protein
sequences of all zyg-11 homologs in C. elegans, C. briggsae, C. remanei, and C.
japonica. As in Figure 2-13 A, full-length protein sequences of all genes were used,
except for the three proteins containing predicted transmembrane domains, ZEEL-1,
Y71A12B.17, and Y55F3C.9. For these three proteins, highlighted with a shaded
pink rectangle, the predicted transmembrane domains were excluded. Y71A12B.17
and the other proteins encoded by genes located in the tandem array are outlined with
a pink dashed box. The frameshift in Y71A 12B. 18 was corrected prior to analysis.
Scale bar indicates amino acid substitutions per sitd. This value is highly deflated
from its true value because the sequence alignment was heavily trimmed prior to
constructing the phylogeny. Values on branches indicate percent bootstrap support.
55
Figure 2-14: (B) Alignment of the amino acid sequences of the transmembrane domains of ZEEL-1 and Y71A12B.17. Sequences were aligned using MUSCLE [35],
using default settings. Colors indicate amino acid classification: hydrophobic, including aromatic (black); acidic or basic (pink); and other (blue). Symbols below
alignment indicate conservation. Above the alignment, horizontal bars indicate predicted transmembrane helices for ZEEL-1 (dark grey) and Y71A12B.17 (light grey).
Predictions were generated using (from top to bottom): TopPred [54], Tmpred [123],
TMHMM [53], SOSUI [112], PHDhtm [137], and HMMTOP [13].
types.
Next, we tested whether heat-shock expression of zeel-1 could rescue the lethality
caused by heat-shock expression of peel-1. We generated five extra-chromosomal
arrays and one single-copy insertion of Phsp-16.41::zeel-1, and we tested these against
one array and one insertion of Phsp-16.41::peel-1. Heat-shock expression of zeel-1
was able to rescue the lethality caused by Phsp-16.41::peel-1, but only when Phsp16.41::peel-1 was expressed from insertions, not arrays (Figure 2-15 B). The ability of
Phsp-16.41::zeel-1 to rescue Phsp-16.41::peel-1 even when both were expressed from
single-copy insertions (Figure 2-15 B) indicates that insofar as these two transgenes
produce equivalent levels of protein, zeel-1 -mediated rescue does not require levels of
ZEEL-1 to be higher than levels of PEEL-1.
Sperm-supplied PEEL-I may act directly during the two-fold stage
The fact that peel-1 -affected embryos do not exhibit defects until late in development
at two-fold stage is surprising because sperm-supplied factors are thought to act
only during egg-activation and first cleavage [77]. One explanation for this paradox
is that the late-occurring defects might be a downstream manifestation of a cryptic
defect earlier in development. Alternatively, sperm-supplied PEEL-i might persist
long after fertilization but only become toxic at the two-fold stage. While we have
been unable to visualize PEEL-i after fertilization, using either PEEL-1::GFP or
the anti-PEEL-i antibody (presumably because PEEL-i becomes too diffuse), three
observations are consistent with PEEL-1 acting directly during the two-fold stage.
First, pre-two-fold embryos were able to develop normally even when exposed
56
A
hs::peel-1
r
""'"" hs::peel-1 insertion (Phsp-16.41)
100
S
75
---
--
-hs::peel-1
insertion (Phsp-16.2)
hs::peel-1 array (Phsp-16.41)
- hs::peel-1 array (Phsp-16.2)
(D
50
25
0
B
2
4
6
8
Time since start of heat-shock (hours)
hs::peel-1 + hs::zeel-1
100 i-
.
V.s.
75
hs::peel-1 alone
0)
50
25
p > 0.05
P 0.05
hs:peel-1 array;
hs::zeel-1
array
p > 0.05
hs::peel-1 array;
hs::zeel-1 insertion
<10-20
p <hs::zeel-1
hs::peel-1 insertion;
array
p 10-20
p0~<
hs::peel-1 insertion;
~hs:-:zeel-1 insertion
0
Time since start of heat-shock (hours)
a
C
111174 80 162 63 87 46 122 34 67
=13840 91 43 11042 11535 07834
41 98
631
22 51 23 53
9938 11 117
8 35
58 23
F'
100
hs::peel-1 array;
zee-IA/A
32
.02
75
hs::peel-1 insertion;
zeel-1A/A
6
hs::peel-I insertion;
zeel-1+/+
50
hs::zeel-i insertion;
peel-I-affected, male-sired
25
hs::zeel-I array;
peel-1I-affected maie-sired
3-3.5h
3.5-4h
4-4.5h
4.5-5h
5-5.5h
5.5-h
6-.5h
6.5-7h
7-7.5h
Age at start of heat-shock (hours post 4-cell)
Figure 2-15: Ectopic expression of peel-1 and zeel-1 replicates peel-1mediated toxicity and zeel-1-mediated rescue.
(A) Survival curves for heat-shocked adult animals carrying extra-chromosomal arrays
or single-copy genomic insertions of Phsp-16.2::peel-1 or Phsp-16.41::peel-1. Time
zero indicates the start of a one-hour heat-shock at 34C. Curves represent one array and one insertion of Phsp-16.2::peel-1 and two independent arrays and seven
independent insertions of Phsp-16.41::peel-1. Log-rank tests were used to compare i)
insertions v.s. arrays, and ii) Phsp-16.2::peel-1 v.s. Phsp-16.41::peel-1. Data for independent arrays or insertions of Phsp-16.41::peel-1 were combined prior to analysis.
*, p < 10-16. n/s, p > 0.05. n = 40-70 animals
per curve.
57
Figure 2-15:
(B) One array and one insertion of Phsp-16.41::peel-1 were chosen from (A) to
be tested against five independent arrays and one single-copy insertion of Phsp16.41::zeel-1. Animals carrying both types of transgenes were heat-shocked as in
(A). Assays were truncated at 10 hours post-heat-shock. Log-rank tests were used to
compare each assay to the corresponding assay of Phsp-16.41::peel-1 alone from (A)
(p-values shown). n = 45-180 animals per curve.
(C) The following classes of embryos, aged three to 7.5 hours post-4-cell stage, were
heat-shocked for 20 minutes at 34C: (i) zeel-1 (A) embryos carrying a Phsp-16.41::peel1 array (pale green bars); (ii) zeel-1(A) and zeel-1(+) embryos carrying a Phsp16.41::peel-1 insertion (black and grey bars, respectively); and (iii) peel-I-affected,
male-sired embryos carrying either an array or an insertion of Phsp-16.41::zeel-1 (yellow and red bars, respectively). For each genotypic class, the proportion of embryos
arresting at the two-fold stage relative to all embryos that elongated to the two-fold
stage is plotted. Differences between the black and grey bars are not significant (p
0.05, x2 tests for each age class). See Figure 2-16 for the full dataset.
(D-E) Vulva regions of animals carrying an array of Pexp-3::peel-1 and an integrated
copy of Pmyo-3::GFP, a marker of the egg-laying muscles. Somatic inheritance of
the Pexp-3::peel-1 array was followed by co-injection markers, Prab-3::mCherryand
Pmyo-3::mCherry, which express in neurons and the egg-laying and body-wall muscles, respectively. In (D), the egg-laying muscles (arrows) are morphologically normal
and have not inherited the Pexp-3::peel-1 array, as indicated by absence of mCherry
expression. In (E), the left-hand egg-laying muscle is severely atrophied (arrow)
and the right-hand egg-laying muscle is absent. The atrophied muscle cell expresses
mCherry, indicating this cell has inherited the array of Pexp-3::peel-1. In both images,
mCherry expression is also visible in body-wall muscles and neighboring neurons.
(F) Dead GABA neuron (arrow) in an animal carrying an array of Punc-47::peel-1.
The surrounding tissue, including a neighboring, non-GABA neuron (arrowhead) is
morphologically normal.
58
A
60v
( iii h s
el
s r
n z
1
A
(iii)hs:peel-1 insertion,
z el- A
63 46 34 41 12223 8
140
1011380
K
9
058
(iv)hs peel- insertionzel- +1+
1 58
141 1115
LMN
Figure 2-16: Ectopic expression of peel-i and zeel-1.
(A) The full dataset used to generate the plot in Figure 2-15 C is shown. As described in Figure 2-15 C, the following classes of embryos, aged three to 7.5 hours
post-4-cell stage, were heat-shocked for 20 minutes at 34C: (i) wild-type (ii) zeel1 (A)/zeel-1(ZA) embryos carrying a Phsp-i16.41i::peel-i array (iii) zeel-1(/A)/zeel-1(zA)
embryos carrying a Phsp-6.4i::peel-1insertion (iv) zeel-i(+)/zeel-i(+) embryos car-
rying a Phsp-16.41::peel-1 insertion (v) peel-i1-affected, male-sired embryos carrying
a Phsp-16.4i::zeel-1 array (vi) peel-i-affected, male-sired embryos carrying a Phsp-
16.41i::zeel-i insertion Each embryo was classified as hatching (white) or arresting
before the two-fold stage (black), at the two-fold stage (dark grey), or after the twofold stage (light grey). Numbers above bars indicate the total number of embryos in
each age class.
(B-J) Time series images of heat-shocked, zeel-1(zA)/zeel-1(zA) embryos carrying either an insertion (B-D) or an array (E-J) of Phsp-i6.4i::peel-i. When visible, epidermal leakage and excretory cell distention are labeled. In (G, J), tails are indicated to
help orient the viewer.
(K-M) Images of heat-shocked, adult hermaphrodites carrying an insertion of Phsp16.41::peel-1. Animals were imaged shortly after paralysis had begun. Necrosis is
visible in the head (K) and gonad (M), and the excretory cell is distended (L).
(N-O) Images of animals carrying an array of Pexp-3::peel-1 and an integrated copy
of Pmyo-3::GFP,which serves as a marker of the anal depressor muscle. Intestinal
bloating is visible in both animals, but only in (N) is the anal depressor muscle absent.
59
to more PEEL-1 protein than is delivered by sperm. We heat-shocked pre-two-fold
embryos, aged three to 7.5 hours after the 4-cell stage, carrying either an array or
insertion of Phsp-16.41::peel-1. Embryos were heat-shocked for 20 minutes at 34C.
Longer and earlier heat-shock were not possible because even in wild-type embryos,
such conditions cause premature arrest (personal observations). Except for occasional
subtle shape defects during early elongation, more than 97% of array- and insertioncarrying embryos developed normally to the two-fold stage (n > 200; Figure 2-16 A).
This result is consistent with sperm-supplied PEEL-1 being able to persist until the
two-fold stage without manifesting a visible phenotype earlier in development.
Second, heat-shock expression of peel-1 as late as 30 minutes before the twofold stage phenocopied the defects observed in peel-1-affected embryos. In the heatshocked embryos described above, embryos carrying an array of Phsp-16.41::peel-i
uniformly arrested at the two-fold stage and showed muscle detachment, epidermal
leakage, and excretory cell distention (Figure 2-15 C, Figure 2-16 B-J). These defects
were also observed among insertion-carrying embryos, although their occurrence required earlier induction of the transgene (Figure 2-15 C). In addition, consistent with
heat-shock treatment exposing embryos to more PEEL-1 protein than is delivered by
sperm, the defects induced by heat-shock were often more severe than those observed
in peel-1-affected embryos, and even among insertion-carrying embryos, the defects
could not be rescued by endogenous expression of zeel-i (Figure 2-15 C). These results demonstrate that as long as peel-i is expressed at or just before the two-fold
stage, presence of PEEL-1 in the early embryo is dispensable for the two-fold arrest.
Finally, rescue of peel-1 -affected embryos did not require early expression of zeel-1.
We induced zeel-i expression in male-sired, peel-i-affected embryos by heat-shocking
embryos carrying either an array or an insertion of Phsp-16.41::zeel-1. Heat-shock
treatment rescued 53-100% of array-carrying embryos (n = 17-43), as long as heatshock treatment occurred at least one hour before the two-fold stage (Figure 2-15
C). (Here rescue is defined as elongation past the two-fold stage. See Figure 2-16
A for the proportion of embryos that hatched.)
Similar results were observed for
insertion-carrying embryos, although rescue activity required earlier induction of the
60
transgene (Figure 2-15 C). These results imply that ZEEL-1 can neutralize spermsupplied PEEL-1 at any time before the two-fold stage. This scenario is temporally
discordant with PEEL-1 causing a cryptic defect early in development.
subsectionCell-specific expression of peel-i produces cell-specific ablation The test
the cell-autonomy of peel-i killing, as well as the utility of peel-1 as a tool for cellspecific ablation, we expressed peel-I under the control of each of two cell-specific promoters: Punc-4 7, which expresses in the GABA-ergic neurons
[83];
and Pexp-3, which
expresses in the egg-laying muscles and the anal depressor muscle (C. Frokjr-Jensen,
personal communication). For each promoter construct, we examined the presence
or absence of the corresponding cell types for four independent extra-chromosomal
arrays.
In both muscle cells and neurons, cell-specific expression of peel-1 produced cellspecific ablation, although the efficacy of ablation varied among arrays. Three of
the Punc-47::peel-1 arrays killed 94.2-99.8% (n= 241-453) of GABA-ergic neurons,
although the fourth array killed only 28.6% (n = 350) of them. Each of the Pexp3::peel-i arrays killed 6-27% (n = 83-90) of anal depressor muscles and 74-94% (n
= 140-180) of egg-laying muscles. Of the egg-laying muscles that remained alive, all
were severely atrophied (Figure 2-15 E). Lower toxicity to the anal depressor muscle
may have been caused by selection bias among our arrays, because animals lacking
the anal depressor muscle were very severely constipated and therefore more slowgrowing than others. In addition, even among animals in which the anal depressor
remained alive, constipation was prevalent (Figure 2-17), indicating that function of
this muscle was impaired.
Aside from the defects caused by ablation of the corresponding cell types, animals
carrying either type of construct were morphologically and behaviorally normal, consistent with PEEL-1 acting cell-autonomously. In addition, with one exception, no
defects were observed outside the ablated cells types (Figure 2-15 F). The exception
was that the three high kill Punc-47::peel-1 arrays were lethal to the animal when
inherited somatically along the lineage of the four RME neurons: for these three arrays, embryo and early larval lethality was very high (33.0-57.7%; n = 327-1095), and
61
the only animals surviving to adulthood were those that lost the arrays somatically
in the four RME neurons. Given that the RME neurons are not required for survival
[82], this lethality implies that either (i) expression of peel-i in the RME neurons kills
a neighboring cell nonautonomously; or (ii) expression of peel-i is leaky and kills one
or more essential cells along the RME lineage. While we did not distinguish between
these possibilities, we note that the sister cell to one RME neuron is the excretory
cell, which is essential for survival.
2.2.2
Materials and Methods
Strains
Strains were maintained at 19-23C on NGM plates spotted with E. coli strain OP50.
In all age-effect experiments, strains were strictly maintained at 20C. For all transgenes described in this publication, only one array and/or one insertion is given in
the strain list, although unless otherwise specified in the Results subsection, multiple
independent arrays or insertions were examined.
CB4088 him-5(eI 90) V.
CB4856 Hawaii natural isolate carrying niDf9 I. niDf9 designates the
19 kb deficiency spanning peel-1 and zeel-1.
EG1285 oxIs12Punc-47.:GFP;lin-15(+)j lin-15B(n765) X.
EG4322 ttTi5605 II; unc-119(ed3) IIl.
EG4348 Utah natural isolate carrying peel-1(qq99) I. EG4348 was collected by M. Ailion from Salt Lake City, Utah (this publication).
qq99
designates the naturally-occurring nonsense mutation in peel-1.
EG5389 qqlr7[peel-1(qq99)j I; oxIs494[Ppeel-1::GFP]II; unc-119(ed3) III.
EG5655 qqlr7[peel-1(qq99)] I; oxSil 9[peel-1 (+), Cbr-unc-119(+)] II; unc-119(ed3)
EG5766 qqlr7[peel-1(qq99)] I; oxSi77fPpeel-1::peel-1::GFPjII; unc-119(ed3) III.
EG5801 oxSi87[Ppeel-1::peel-112a.a.::GFP]
II; unc-119(ed3) III.
EG5955 qqlr7[peel-1(qq99)] I; tt Ti5605 II; unc-119(ed3) III; oxEx1462[Phsp-16.41::peel62
A
Anal depressor muscle
Dead/Absent
Alive
Inherited
% Killing
(total cells inheriting % Constipated
the array)
(total animals)*'
Did not inherit
araPexp-peelI Pexp-3:peel-
1
6
84
10
6.7% (90)
87% (90)
3
5
78
17
6.0% (83)
89% (83)
Calculated among animals inwhich the array had been transmitted to the anal depressors muscle
B
Egg-laying muscles
Dead/Absent
Alive
Morphologically
normal
Atrophied
Array
Inherited
Pexp-3peel-I
arrav
Did not inherit
Pexp. -peel-I
array
11
0
0
36
0
0
1169
-p
3
104
Inherited
Did not inherit
Pexp-:peel-I Pexp-3::peelarray
array
% Killing
(total cells inheriting
% Egg-laying
defective
the arrav)
(total animals)*
20
93.4% (180)
1000% (930)
60
74.3% (140)
100% (70)
y
Calculated among animals inwhich the array had been transmitted to the egg-laying muscles
C
GABA neurons
Dead/Absent
Arrav
rray
Alive
Inherited
Did not inherit
Punc-47::peel-1
array
Punc-47::peel-I
array
% Killing
(total cells inheriting % Embryo/larva arrest
the array)
(total)
% Mosaics
(total adults)
1
452
1
163
99.8% (453)
57.7% (1095)
100% (74)
3
227
14
273
94.2% (241)
33.0% (587)
100%(73)
Figure 2-17: Cell-specific killing via ectopic expression of peel-1.
(A-B) The Pexp-3::peel-1 arrays were crossed to a strain carrying an insertion of
Pmyo-3::GFP,which serves as a marker of the egg-laying muscles and the anal depressor muscle. Live muscle cells were classified as inheriting the array if they expressed the co-injection marker, Pmyo-3::mCherry. One hundred animals were scored
for each array, and two of the four egg-laying muscles were scored per animal.
(C) The Punc-47::peel-1 arrays were crossed to a strain carrying an insertion of
Punc-47::GFP,which serves as a marker for the GABA neurons. Live neurons were
classified as inheriting the array if they expressed the co-injection marker, Prab3::mCherry. For each array, 50-74 animals were scored, and six to ten neurons were
scored per animal.
63
1, Cbr- unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
EG5958 qqlr7[peel-1 (qq99)] I; oxSi186(Phsp-16.41::peel-1, Cbr-unc-119(+)]II; unc-
119(ed3) III.
EG5960 qqlr7[peel-1(qq99)] I; oxSi188[Phsp-16.2::peel-1, Cbr-unc-119(+)]II; unc119(ed3) III.
EG5961 qqlr7[peel-1(qq99)] I; tt Ti5605 II; unc-119(ed3) III; oxExl4 64[Phsp-16.2::peel1, Cbr- unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
EG6297 qqlr5/niDf9] I; oxSi298[Phsp-16.41::zeel-1::tagRFP,Cbr-unc-119(+)]II;
unc-119(ed3) III.
EG6298 qqlr5/niDf9] I; tt Ti5605 II; unc-119(ed3) III; oxEx1501[Phsp-16.41::zeel1::tagRFP, Cbr- unc-119(+), Pmyo-2::GFP].
EG6301 qqlr5[niDf9] I; ttTi5605 II; unc-119(ed3) III; oxEx1504[Pexp-3::peel-1,
Cbr-unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
EG6306 qqlr5/niDf9] I; tTi5605 II; unc-119(ed3) III; oxEx1509[Punc-47::peel-1,
Cbr-unc-119(+), Prab-3::mCherry].
MT1344 bli-3(e767) lin-17(n677) I.
MT3301 fem-1(hc17) IV; him-5(e1490) V.
MY19 German natural isolate carrying peel-1(qq98) I. MY19 was collected from Roxel, Germany [51]. qq98 designates the naturally-occurring
nonsense mutation in peel-1.
N2 Laboratory reference strain, Bristol.
PD4790 mIs12[myo-2::GFP,pes-10::GFPand gut::GFP].
QX1015 niDf9 I; qqlr8[N2 =g CB4856, unc-119(ed3)] III.
QX1197 qqlr5[CB4856 = N2, niDf9] I. qqIr5 is an 140-370 kb introgression from
CB4856 into N2. This strain was used in some experiments instead of CB4856, in
order to reduce the genetic variation segregating in the background.
QX1257 niDf9 I; qqlr8/unc-119(ed3)] III unc-119(ed3) III; qqIs2[zeel - lgenomic::GFP,
unc- 119(+)].
QX1264 niDf9 I; qqlr8[unc-119(ed3)]III unc-119(ed3) III; qqEx2[zeel unc-119(+)].
64
1
genomic::GFP,
QX1319 zeel-1(tm3419)/hT2[q~s48j I; +/hT2[qIs48] III.
QX1320 qqIr6[EG4348=gN2, peel-1(qq99)j I; unc-119(ed3) III.
QX1384 niDf9 I; qqIr8[unc-119(ed3)jIII unc-119(ed3) III; qqEx6[Pzeel-1:: zeel -
1
SOL,
unc-119(+)j.
QX1392 qqIr6[peel-1(qq99)] I;unc-119(ed3) III; qqEx3[peel-1(+), unc-119(+)].
QX1409 qqlr7[EG4348=gN2, peel-1(qq99)] I; ttTi5605 II; unc-119(ed3) III.
QX1577 qqIr5[niDf9j I;qqEx1[Pzeel-1::zeel - 1cDNA::GFP, Pmyo-2::RFP].
QX1589 qqIr5[niDf9]I; qqEx4[Pzeel-1::Y71A12B. 17TM:: zeel -
1
SOL,
Pmyo-2::RFP.
QX1605 qqIr5[niiDf9] I; ttTi5605 II; unc-119(ed3) III.
QX1607 qqIr5[niDf9] I; qqEx5[Pzeel-i:: zeel-iTM, Pmyo-2::RFP.
QX1618 qqIr5[niiDf9] I; qqEx7[Plin-26::zeel-1, Pmyo-2::RFP.
QX1619 qqIr5[niDf9] I; qqEx8[Phlh-1::zeel-1, Pmyo-2::RFP.
QX1624 qqlr5[niDf9] I; oxSi186[Phsp-16.41::peel-1, Cbr-unc-119(+)I II; unc-119(ed3)
II I.
QX1650 oxSi19[peel-1(+), Cbr-unc-119(+)j II.
QX1772 qqIr5[niDf9j I;ttTi5605 II; unc-119(ed3) III; oxEx1462[Phsp-16.41::peel1, Cbr-unc-119(+), Pmyo-2::mCherry, Pmyo-3::mCherry, Prab-3::mCherry].
SJ4157 zcls2l[Phsp-16::cpp-1(WT)::3xmyc-His tag + Pmyo-3::GFP]V.
Scoring embryo lethality
In all experiments except the age-effect experiment, embryo lethality from self-fertilizing
hermaphrodites was scored by isolating hermaphrodites at the L4 stage and singling them to fresh plates the following day. After laying eggs for 8-10 hours, the
hermaphrodites were removed and embryos were counted. Unhatched embryos were
counted -24 hours later. To score embryo lethality from mated hermaphrodites,
three or four L4 hermaphrodites were mated to six to ten L4 or young adult males for
24-36 hours. Hermaphrodites were then singled to fresh plates and embryo lethality
was scored as above. Broods were examined for the presence of males 2-3 days later,
and any broods lacking males were excluded. To allow male sperm to age within the
reproductive tract of the hermaphrodite, mated hermaphrodites were removed from
65
males, and lethality was scored among embryos laid three days after removal.
In the age-effect experiment, 91 zeel-1(tm3419)peel-1(+)/ niDf9 hermaphrodites
were singled at the L4 stage and transferred every 12 hours to fresh plates. Hermaphrodites
were discarded after the first 12-hour period in which they failed to lay fertilized embryos. Total embryos were counted at the end of each laying period, and unhatched
embryos were counted -24 hours after each laying period had ended.
To score embryo lethality from partially mated hermaphrodites, 130 zeel-1 (tm3419)peel1(+)/niDf9 hermaphrodites were mated at the L4 stage to an equal number of
PD4790 males, which carry an insertion of the fluorescent marker, Pmyo-2::GFP.
After 24 hours, hermaphrodites were singled and transferred every 12 hours to fresh
plates until day five. Embryo lethality was scored as above, except that after unhatched embryos were counted, hatched and unhatched progeny were classified as
self- or cross-progeny according to presence of pharyngeal GFP. Hermaphrodites laying 100% self-progeny or more than 95% cross-progeny were excluded. The remaining
hermaphrodites, which we define as partially mated, laid -10-50% self-progeny. In
these broods, we calculated the portion of self-progeny, laid during days three to five,
that failed to hatch.
Mapping peel-1 mutations in MY19 and EG4348
Absence of the paternal-effect in EG4348 was mapped relative to bli-3(e767), a visible marker located -10 cM from the peel-i interval. Mapping was performed as described [115]. Briefly, EG4348 males were crossed to MT1344 hermaphrodites, and F1
hermaphrodites were mated to CB4856 males. The resulting hermaphrodite progeny
were allowed to self-fertilize, and their broods were scored for embryo lethality (i.e.
presence of peel-i activity) and presence of Bli animals. Directionality with respect
to bli-3 could be inferred because bli-3 is located at the left-hand tip of chromosome
I.
Preliminary sequence analysis of the peel-i interval in EG4348 was performed by
genotyping EG4348 with a subset of the markers listed in Table S2 of [115]. These
markers tile across the peel-i interval, and they distinguish all haplotypes carrying an
66
intact copy of the peel-i /zeel-i element from all haplotypes lacking it [115]. In other
words, the Bristol-like alleles of these markers are in perfect linkage disequilibrium
with presence of the peel-i /zeel-i element. At all markers we assayed, EG4348 carried
the Bristol-like allele.
Fine-mapping in MY19 and EG4348 was performed by crossing each strain to
MT1344 and collecting Lin Non-Bli and Bli Non-Lin recombinants in the F2 generation. Recombinant animals were genotyped (via a portion of their F3 broods) at each
of two markers flanking the peel-i interval. The right-hand marker for the MY19
cross was a BstCI snip-SNP amplified with primers 5'-GTA TTC CGA CGA TTC
GGA TG-3' and 5'-CAT TGA GAA CAC AAA AAC AAA CG-3'. The right-hand
marker for EG4348 cross was an Afel snip-SNP amplified with primers 5'- GAC ATA
TTT CCC GCA ACC TG-3' and 5'- GTG ACG AGG CTT GAG GAT TC-3'. The
left-hand marker for both crosses was a BanI snip-SNP amplified with primers 5'CGC CAA ATA TGT TGT GCA GT-3' and 5'-CAC CAC GTG TCC TTT CTC
ATT-3'.
Recombinants breaking within the peel-i interval were homozygosed for the recombinant chromosome, and the resulting homozygotes were phenotyped for peel-i
activity. Phenotyping was performed by crossing each line to CB4856 and scoring embryo lethality from self-fertilizing, F1 hermaphrodites and from F1 males backcrossed
to CB4856 hermaphrodites. Recombinants were classified as having peel-1 activity
if these crosses produced ~25% and -50% embryo lethality, respectively. Next, the
locations of recombination breakpoints were mapped more finely by sequencing six
to ten sequence polymorphisms, located throughout the peel-i interval, that distinguish MY19 or EG4348 from Bristol. The MY19 polymorphisms were determined
from the MY19 sequence described in [115], and the EG4348 polymorphisms were
determined by amplifying and sequencing arbitrary fragments from this strain. Some
polymorphisms are shared between MY19 and EG4348, and these were used in both
crosses. For the most informative recombinants, we later sequenced across the entire
breakpoint region in order to map these breakpoints to the level of adjacent polymorphisms. This approach mapped the peel-1 -disrupting mutations to regions of 5 kb in
67
MY19 and 8 kb in EG4348. These intervals were then sequenced in the corresponding
strains, and all sequence polymorphisms were identified. Finally, we genotyped these
polymorphisms in a panel of 38 wild strains previously identified as having intact
peel-i activity [115]. These strains, as well as the primers used for genotyping them,
are given in Figure S2. The MY19 sequence was deposited in GenBank previously
[115], and the EG4348 sequence was deposited under accession number HQ291558.
Identification of peel-1 transcript
RNA was collected from mixed-staged Bristol animals by freeze-cracking and extracting in Trizol (Invitrogen) according to the manufacturers protocol.
Reverse
transcription-PCR (RT-PCR) was performed using pairs of primers flanking each
candidate mutation in MY19 and EG4348. For each pair of primers, the forward and
reverse primers were located ~100 bp apart, and two reactions were performed, one
using each of the two primers as the RT primer. Product was observed for only one
pair of primers, and for that pair, only in one direction. These primers were 5'-ACA
TGT ATC TTG ATC TGC CTG A-3' (forward) and 5'-AAA AAT TAA CCA CAA
TGA AGC AA-3' (reverse), and product was only observed using the reverse primer
as the RT primer. To recover the remainder of this putative transcript, 3' and 5'
RACE were performed using standard methods [55]. For 3' RACE, the RT reaction
was performed using 5'-GTT TTC CCA GTC ACG ACT TTT TTT TTT TTT TTT
TT-3', and PCR was performed using the gene-specific primer, 5'-ACA TGT ATC
TTG ATC TGC CTG A-3' (forward) and the adaptor primer, 5'-GTT TTC CCA
GTC ACG AC-3' (reverse). For 5' RACE, the RT reaction was performed using a
gene-specific primer that spanned the putative stop codon, 5'-TCA ATT TCA TGG
ATT TTC AAC A-3', and PCR was performed using 5'-GGC CAC GCG TCG ACT
AGT ACG GGI IGG GII GGG IIG-3' (forward) and a nested, gene-specific primer,
5'-AAA AAT TAA CCA CAA TGA AGC AA-3' (reverse). Then, a second round
of PCR was performed using the adaptor primer, 5'-GGC CAC GCG TCG ACT
AGT AC-3' (forward) and another nested, gene-specific primer, 5'-AGA GCA ATA
ACA TGC GCA AA-3' (reverse). SuperScript III (Invitrogen) was used in all RT
68
reactions, and PlatinumTaq (Invitrogen) was used for all PCR reactions. The peel-i
transcript did not contain a splice leader sequence and was deposited in GenBank
under accession number HQ291556. More recently, the peel-i transcript was identified independently by WormBase curators and assigned the identification number,
Y39G1 OAR. 25.
To search for transcripts carrying both peel-i and zeel-1, an RT reaction was
performed using the peel-i-specific primer, 5'-AAA AAT TAA CCA CAA TGA AGC
AA-3', and PCR was performed using a forward primer located in the 3' end of zeel-i
(5'-CCA TCC GAG ATA ACC GAA AA-3') and a reverse primer located in the 5'
end of peel-i (5'-AGA GCA ATA ACA TGC GCA AA-3'). No product was observed.
Quantitative RT-PCR
CB4088 and MT3301 animals were grow at 15C and synchronized at the LI stage
by bleaching and hatching overnight in M9. Ls were split into two populations,
and one population was shifted to 25C. When animals had reached young adulthood, hermaphrodites and males were separated by hand, and RNA was collected
as above. Real-time PCR of peel-1, spe-9, and rpl-26 was performed in triplicate,
for 40 cycles, on an ABI 7900HT using the QuantiTect SYBR Green Kit (Qiagen).
Relative expression levels of peel-i and spe-9 were calculated separately for males
and hermaphrodites, using the 2 AAC' method, with rpl-26 as the endogenous control
and the 15C MT3301 sample as the reference sample. Primers used to amplify peel-i
were 5'-TAC ACC CGT CAC ACC AAC TG-3' and 5'-TCC GAC TAT GAT GTT
CCA CAA-3'; primers for spe-9 were 5'-CGG CTT GCA TAC ACA ATG AG-3' and
5'-ACG CCA TGA CTC TTG CTC TT-3'; and primers for rpl-26 were 5'-TCC AAT
CAG AAC CGA TGA TG-3' and 5'-GTG CAC AGT GGA TCC GTT AG-3'.
Among the hermaphrodite samples, relative expression levels of peel-i and spe-9
were roughly equivalent, except for the 25C MT3301 sample, where expression of
peel-i and spe-9 was undetectable. That is, in this sample, signal for peel-i and
spe-9 failed to rise above the detection threshold, even after 40 cycles, despite rpl-26
amplifying normally.
69
Single-molecule FISH
Single molecule FISH of was performed as in [105], with the embryos and hermaphrodites
squashed down to ~9 pm thickness for imaging. Automated counting of nuclei in embryos was performed using software developed in [105, 104].
Rescue of peel-1
Transgenic animals carrying peel- I(+) were generated by two methods: bombardment
[101] and Mos1-mediated, single-copy insertion [43]. For bombardment, a fragment
containing the Bristol allele of peel-1, along with -2.8 kb of upstream sequence and
-1 kb of downstream sequence, was excised from fosmid WRM0633bEO9 (Bioscience
LifeSciences, Nottingham, UK) using AhdI and NgoMIV. This fragment was cloned
into the yeast shuttle vector, pRS246 (ATCC, Manassas, VA), via yeast-mediated
ligation [106] of the fragments ends. The resulting plasmid, pHS11, was bombarded
into QX1320, along with the unc-119(+) rescue vector, pDP#MM016B [81]. Bombardment was performed as in [88], although only extra-chromosomal arrays were recovered. Nine independent transgenic lines were tested for peel-1 activity by crossing
them to CB4856 and scoring embryo lethality from self-fertilizing, F1 hermaphrodites
(self-cross) and F1 males backcrossed to CB4856 hermaphrodites (backcross).
For Mos1-mediated insertion, the peel-1 fragment from pHS11 was amplified by
PCR, using primers having NheI cut sites, and this amplicon was cut with NheI and
ligated into pCFJ151 [43] linearized with AvrII. The resulting plasmid, pHS26, was
injected into QX1409 along with the vectors needed to generate single-copy insertions [43]. Insertion-carrying animals were recovered by the direct insertion method
[43], and five independent insertion-carrying lines were tested for peel-i activity as
above. For one of the two insertions that did exhibit peel-i activity, the self-cross
and backcross were repeated, and hatched progeny were collected and genotyped for
a PCR-length polymorphism located less than 1 kb from niiDf9. The primers used to
amplify this polymorphism were 5'-TGG ATA CGA TTC GAG CTT CC-3' (forward)
and 5'-CCC CCT AAT TTC CAA GTG GT-3' (reverse).
70
For three of the peel-i array lines, a small number of severely deformed Lis were
observed in the backcross, similar to the escapers typically observed among peel-1affected embryos sired by hermaphrodites. We suspected that these Ls had escaped
the paternal-effect due to partial germline silencing of the peel-i arrays. Consistent
with this hypothesis, we genotyped 13 of these animals, using the PCR-length polymorphism described above, and all were zeel-i (niDf9) homozygotes. We then calculated the frequency of these escapers relative to the total number of peel-i-affected
progeny (i.e. relative to the total number of dead embryos and deformed Lis).
ZEEL-1::GFP fusion and domain swapping
ZEEL-1::GFP was generated by amplifying GFP from PD95.75 and inserting it into
pHS4.1, a genomic subclone of zeel-1(+) described previously [115].
pHS4.1 was
linearized with AhdI, and yeast-mediated ligation [106] was used to insert GFP just
upstream of the zeel-i stop codon. Later, a second ZEEL-i::GFP construct was
generated using the cDNA of zeel-1, instead of the genomic locus. This construct
was generated by first cutting pHS4.1 with EcoNI and BglII, in order to remove the
entire coding region of zeel-1, and then inserting a full cDNA of zeel-1, followed by
GFP. The cDNA of zeel-i was cloned previously [115], and this replacement was
performed using yeast-mediated ligation [106]. Both constructs showed full rescue of
peel-i-affected embryos, and data from the two constructs were combined.
To generate ZEEL -
1
SOL,
pHS4.1 was cut with EcoNI and KpnI, and the frag-
ment containing zeel-i codons 5 to 205 was removed. The remaining fragment was
then re-circularized, using yeast-mediated ligation [106], to fuse codon 4 to codon
206. To generate ZEEL
-
1
TM,
the entire coding region of zeel-i was excised from
pHS4.1 using EcoNI and BglII, and this fragment was replaced with a partial cDNA
of zeel-i encoding the first 205 amino acids of the protein. This replacement was
performed using yeast-mediated ligation [106].
To generate Y71A12B.17TM::ZEEL - 1SoL, the coding region of zeel-i was ex-
cised from pHS4.1, as above, and yeast mediated ligation [106] was used to replace
this fragment with a partial cDNA of Y71A12B.17, followed by a partial cDNA of
71
zeel-1. The resulting construct contained the N-terminal 207 codons of Y71A12B.17
fused to the C-terminal 712 codons of zeel-1. The junction of this fusion was chosen
to overlap a string of seven amino acids (KNERKEG) that are perfectly conserved
between the two proteins. The Y71A12B.17 cDNA was cloned by reverse transcribing RNA from the Bristol strain using primer 5'-TTG AAC AAA AAC AAT GGA
TAT GTA A-3', and then performing PCR using primers 5'-GGG GAC AAG TTT
GTA CAA AAA AGC AGG CTT CAT GTC GGA TTT CGA CTC AGA-3' (forward) and 5'-GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC ATT TAT
TAA CTC CAA CAA TGA TTC G-3' (reverse). This PCR product was then cloned
into the vector, pDONR221 (Invitrogen), using the Gateway cloning kit (Invitrogen).
The Y71A 12B. 17 cDNA differs slightly from the WormBase gene prediction and was
deposited in GenBank under accession number HQ291557.
All constructs were bombarded [88] into QX1015 along with the unc-119(+) rescue vector, pDP#MM016B [81], or they were injected [39] into QX1197 at ~80ng/pl,
along with the fluorescent marker, Pmyo-2::RFP at 3 ng/pl. To test each transgene for its ability to rescue peel-i-affected embryos, transgenic animals were crossed
to the Bristol strain, and lethality was scored among embryos derived from two
crosses: self-fertilizing F1 hermaphrodites (self-cross); and F1 males backcrossed to
hermaphrodites of the original transgenic line. To calculate hatch rates among peel-I affected embryos inheriting ZEEL-1::GFPand ZEEL-lTM, transgenic animals were
crossed to QX1319, and embryos were collected from: (i) transgenic, self-fertilizing,
F1, zeel-1(tm3419)pee-1(+)/niDf9 hermaphrodites; and (ii) transgenic, F1, zeel1(tm3419)peel-1(+)/ niDf9 males backcrossed to non-transgenic, niDf9/rniDf9 hermaphrodites.
Inheritance of ZEEL-1::GFP and ZEEL - lTM was inferred by expression of the coinjection marker, Pmyo-2::RFP,which can be scored even in arrested embryos.
Other transgenes
All other transgenes were generated using the three-site Gateway system from Invitrogen. This method allows three separate DNA fragments to be joined together and
inserted into pCFJ150, which contains Cbr-unc-119(+) and the sequences needed for
72
Mos1-mediated insertion at the ttTi5605 Mos site on chromosome II [43]. In most
cases, this method was used to join together a promoter of interest, a coding sequence,
and a 3' UTR.
Ppeel-1::GFP, Ppeel-1::PEEL-112a.a. ::GFP, and the PEEL-1::GFP fusion
For Ppeel-1::GFP,we joined together the peel-1 promoter, GFP, and the peel-i
3'UTR. For the peel-1 promoter, we used all intergenic sequence between the peel-i
start codon and last coding segment of zeel-1 (i.e. 2473 bp of sequence). The peel-i
3' UTR was determined empirically and extended 86 bp downstream of the peel-i
stop codon. For GFP, we used a variant containing S65C and three internal introns
(identical to the variant in pPD95.75).
For Ppeel-i::PEEL-
112a.a. ::GFP,
the PEEL-1 leader peptide was added by ex-
tending the promoter fragment to include the first 12 amino acids of PEEL-1. This
signal peptide was discovered while we were investigating a possible regulatory role of
the first intron of peel-1. We had generated a GFP reporter driven by the peel-i promoter and the first intron of peel-1, and this construct also happened to carry the first
12 amino acids of PEEL-1. GFP driven by this construct was packaged into sperm
(data not shown), and in order to confirm that sperm packaging was caused by the
leader peptide, rather than the intron, we generated Ppeel-1::PEEL- 112a.a.::GFP
(which excludes the first intron). Conversely, we also generated a reporter carrying
the first intron and a randomized leader peptide, and for this construct, no sperm
packaging was observed (data not shown).
To tag PEEL-1 with GFP, the promoter fragment was extended even further to
include the entire peel-i gene, up to (but excluding) the stop codon.
All three constructs were injected into EG4322 or QX1409, and single-copy insertions were obtained using the direct insertion MosSCI method [43]. Three to six
independent insertions were analyzed for each construct, and no differences were observed among insertions of the same construct. We note that although PEEL-1::GFP
appears to localize normally, it failed to exhibit peel-i activity (data not shown),
presumably because the GFP tag inhibited function.
73
Plin-26::zeel-1 and Phlh-1::zeel-1
For Plin-26::zeel-1 and Phlh-1::zeel-1, the promoters of lin-26 or hlh-1 were joined
to the cDNA of zeel-1 and the 3' UTR of let-858. For the promoters of lin-26 and
hlh-1, 7122 bp of sequence and 3037 bp of sequence upstream of the respective start
codons were used. For the let-858 3' UTR, 434 bp of sequence downstream of the
stop codon was used. Transgenic animals were generated by injecting Plin-26::zeel-1
and Phlh-1::zeel-1 into QX1197 at -80 ng/pl, along with the fluorescent marker,
Pmyo-2::RFP at 3 ng/pl.
To evaluate rescue among male-sired embryos, transgenic animals were crossed to
QX1319, and transgenic, F1, zeel-1 (tm3419)peel-1 (+)/niDf9 males were backcrossed
to non-transgenic, niiDf9/niDf9 hermaphrodites. Embryos were dissected from these
hermaphrodites and imaged every 10-20 minutes, starting before the two-fold stage
and ending at least six hours after the two-fold stage. To evaluate rescue among
hermaphrodite-sired embryos, transgenic lines were crossed to QX1319, and embryo
viability was scored among embryos collected from transgenic, self-fertilizing, F1, zeel1 (tm3419)peel-1 (+)/niDf9 hermaphrodites. Among both male- and hermaphroditesired embryos, inheritance of the transgene was inferred by expression of Pmyo2::RFP.
Heat-shock constructs and constructs for cell-specific expression of peel-1
Phsp-16.41::peel-1, Phsp-16.2::peel-1, Pexp-3::peel-1, and Punc-47::peel-1 were generated by joining the peel-i cDNA downstream of the appropriate promoter and
upstream of the tbb-2 3' UTR. We describe the promoter and 3'UTR fragments
in terms of length of sequence upstream or downstream of the appropriate start
or stop codons: Phsp-16.41 (501 bp), Phsp-16.2 (493 bp), Pexp-3 (2877 bp), and
Punc-47 (1251 bp), and tbb-2 3' UTR (331 bp).
Phsp-16.41::peel-1 and Phsp-
16.2::peel-1 were injected into QX1409 at 25 ng/pl, and arrays and MosSCI insertions
were recovered as in [43]. The arrays carry co-injection markers Pmyo-2:: mCherry,
Pmyo-3:: mCherry, and Prab-3::mCherry. Pexp-3::peel-1 and Punc-47::peel-1 were
74
injected into QX1605 at 25 and 10 ng/pl respectively, along with co-injection markers
Prab-3::mCherry, Pmyo-2:: mCherry, and Pmyo-3::mCherry (for Pexp-3::peel-1) and
marker Prab-3::mCherry(for Punc-47::peel-1). In all cases, Pmyo-3::mCherry and
Prab-3::mCherrywere injected at 10 ng/pl, and Pmyo-2::mCherry was injected at 5
ng/pl.
Phsp-16.41::zeel-1 was generated using the hsp-16.41 promoter described above,
the zeel-i cDNA, and the let-858 3'UTR fused downstream of tagRFP. tagRFP was
added to confirm expression of zeel-i after heat-shock. Phsp-16.41::zeel-i was injected at 10 ng/pl into QX1605 and the arrays and the MosSCI insertion were recovered as in [43], except that a GFP-based co-injection marker (Pmyo-2::GFPinjected
at 2.5 ng/pl) was used in order to distinguish these arrays from the Phsp-16.41::peel-1
arrays.
Microscopy and analysis of live embryos
Imaging of fixed embryos and live imaging'of ZEEL-1::GFPembryos was performed
on a PerkinElmer RS3 spinning disk confocal. All other imaging was performed on a
Nikon 90i equipped with a CoolSNAP HQ2 camera and a X-Cite 120 Series fluorescent
light source. Images were acquired and background subtracted with either Volocity
(PerkinElmer) or NIS Elements (Nikon), and (in some cases) multiple channels were
overlaid in Adobe Photoshop. To image dissected gonads, spermatocytes, and sperm,
adult males or mated hermaphrodites were dissected into sperm media containing
dextrose (50 mM Hepes, 1 mM MgSO 4 , 25 mM KCl, 45 mM NaCl, 5 mM CaCl2 , 10
mM dextrose).
To measure the onset of epidermal leakage in peel-i-affected embryos, pre-arrest
embryos were dissected from the following crosses. Hermaphrodite-sired embryos were
dissected from (i) self-fertilizing, zeel-i (tm3419)peel-1 (+)/niDf9 hermaphrodites; and
(ii) self-fertilizing, zeel-1(tm3419)peel-1(+)/ zeel-1(tm3419)peel-1(+) hermaphrodites.
Male-sired embryos were dissected from niDf9/niDf9 hermaphrodites mated to three
types of males: (i) zeel-1(tm3419)peel-1(+))/niDf9; (ii) zeel-1(tm3419)peel-1(+)/zeel1(+)peel-1(+);and (iii) zeel-1 (tm3419)peel-1 (+)/ zeel- 1 (+)peel-I(+); oxSi 19/peel-1(+)j +.
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The self-fertilizing hermaphrodites were aged 24 hours post-L4 at the time of dissubsection, and mated hermaphrodites were aged 24-48 hours at the time of dissubsection. After dissubsection, embryos were imaged every 10 minutes, starting before the
1.5-fold stage and ending seven or more hours after the 1.5-fold stage. The onset of
epidermal leakage was calculated as the time between the 1.5-fold stage and the first
frame in which leakage was observed. Calculations were truncated at seven hours
past the 1.5-fold stage because this represents one hour after the average hatching
time of wild-type embryos. Finally, embryos were binned into 30-minute intervals in
order to generate the inverted histograms shown in Figure 4A.
To calculate the percentage of peel-i-affected embryos elongating past two-fold,
zeeI-1(tm3419)peel-1(+)/niDf9 hermaphrodites were isolated at the L4 stage and
allowed to age for 24, 48, 60, and 72 hours. Embryos were then dissected and imaged
every 20 or 30 minutes for at least 10 hours.
Fixation of sperm and embryos
Anti-PEEL-i is a rabbit polyclonal generated against the C-terminal 15 amino acids
of PEEL-1. This antibody was generated and purified by GenScript, Piscataway, NJ.
1CB4 is a mouse monoclonal used to stain FB-MOs [96]. 1CB4 was a gift from Steven
L'Hernault. To stain sperm, adult males were dissected into sperm media on charged
slides, freeze-cracked in liquid nitrogen, and fixed overnight in -20C methanol. Slides
were washed with PBST (PBS + 0.1% Triton-X 100), blocked for 30 minutes with
PBST + 0.5% BSA, and incubated for four hours with anti-PEEL-1 (1/100) and
1CB4 (1/2000), diluted in PBST + 0.5% BSA. Slides were then washed three times
in PBST and incubated for two hours with Alexa568-labeled anti-mouse (1/500) and
Alexa488-labeled anti-rabbit (1/500) (Invitrogen), diluted in PBST + 0.5% BSA.
Slides were washed again three times in PBST and mounted in Vectashield mounting
media with DAPI.
To visualize actin filaments in peel-i -affected embryos, embryos were stained with
Alexa568-labeled Phalloidin (Invitrogen) according to Protocol 7 in [120]. To visualize all the other proteins, embryos were stained with monoclonal antibodies MH2
76
(perlecan), DM5.6 (myosin heavy chain A), MH5 (VAB-10A), and MH4 (intermediate filaments). All monoclonals were obtained from The Developmental Studies
Hybridoma Bank, Iowa City, Iowa. For these experiments, embryos were fixed for
10 minutes in 3% paraformaldehdye, freeze cracked in liquid nitrogen, and incubated
for 5-10 minutes in -20C methanol. Embryos were then washed three times in PBST
and incubated overnight with the primary antibody diluted in PBST + 1% BSA.
MH2, MH4, and MH5 were diluted 1/150, and DM5.6 was diluted 1/1000. Embryos
were washed three times in PBST and incubated overnight with Alexa488-labeled
anti-mouse (1/500) (Invitrogen), diluted in PBST + 1% BSA. Embryos were washed
again three times in PBST and mounted in Vectashield mounting media with DAPI.
Phylogenetic analysis of zeel-1
Separate phyolgenetic trees were built for: (i) zyg-11 and all zyg-11 homologs in
C. elegans; and (ii) zyg-11 and all.zyg-11 homologs in C. elegans, C. briggsae, C.
remanei, and C. japonica. zyg-11 homologs were defined as all genes carrying the
zyg-11-like leucine-rich repeat region. After removing the predicted transmembrane
domains of ZEEL-1, Y71A12B.17, and Y55F3C.9, all protein sequences were aligned
using MUSCLE [35]. The alignments were performed using the BLOSUM30 substitution matrix, a gap open penalty of -10, and a gap extend penalty of -1. The C.
elegans-only alignment was trimmed to exclude residues having gaps in more than
90% of sequences, and the multi-species alignment was trimmed using the heuristic
method, automated1, from TrimAL [24], which is optimized for maximum likelihood
tree construction. Finally, phylogenetic trees were constructed using PhyML [49],
using the LG substitution model
[75],
zero invariant sites, and four substitution rate
categories. Branch support was determined using bootstrap sampling with 100 replicates.
Divergence between zeel-1 and Y71A12B.17 was determined by aligning the two
proteins with MUSCLE [35], trimming the alignment of gaps, and using PAML [46]
to calculating synonymous site divergence on the corresponding nucleotide sequences.
(The total length of gaps was less than 0.1% of the length of the total alignment.)
77
The summary statistics are as follows: number of synonymous sites = 715.5; number
of non-synonymous sites
=
2002.5; synonymous substitutions per site (ds)
non-synonymous substitutions per site (dN)
=
=
1.0709;
0.3267.
Heat-shock
Adults and larvae were heat-shocked by submerging sealed, agar plates in a 34C water
bath for one hour. Embryos were heat-shocked by mounting embryos on an agar pad,
incubating the slide at 19-20C for the prescribed number of hours before placing the
slide on the floor of a sealed, 1 cm x 8 cm x 8 cm plastic box, and submerging the
box in a 34C water bath for 20 minutes.
every 20 minutes for at least 10 hours.
After heat-shock, embryos were imaged
Initially, embryos were staged directly by
collecting and mounting 4-cell embryos. Later, when it became clear that the vast
majority of heat-shocked embryos developed to the two-fold stage without defects or
delay, throughput was increased by collecting mixed stage embryos and mounting,
incubating, and heat-shocking as above. These embryos were staged relative to the
time at which they initiated elongation and by comparing their morphology before
heat-shock to images of embryos that had been staged using the direct method.
Cell-specific killing
To quantify peel-1-mediated killing of the egg-laying muscles and the anal depressor
muscle, the Pexp-3::peel-1 arrays were crossed to SJ4157, which carries an integrated
array of the muscle marker, Pmyo-3::GFP. In day-one, F1 hermaphrodites, two of
the four egg-laying muscles and the single anal depressor muscle were observed and
classified as live or dead based on expression of GFP. Live egg-laying muscles were
classified as morphologically normal or atrophied, and both live egg-laying muscles
and live anal depressor muscles were then classified as mCherry+ or mCherryindicating that they had or had not inherited the Pexp-3::peel-1 array. For each cell
type, the percent of cells killed by the arrays was calculated assuming that all dead
cells had inherited the array. In addition, each F1 animal was classified as constipated
if it contained bacteria in the posterior intestine and as egg-laying defective if it
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contained three-fold embryos in the uterus.
To quantify peel-I-mediated killing of GABA neurons, the Punc-47::peel-I arrays
were crossed to EG1285, which carries an integrated array of the GABA-neuron
marker, Punc-47::GFP.GABA-neurons were observed in F1 hermaphrodites, and
peel-1-mediated killing was quantified as above. Typically, the DVB neuron and five
to nine ventral cord neurons were scored per hermaphrodite. In addition, each animal
was classified as mosaic or non-mosaic based on expression of the co-injection marker,
Prab-3::mCherry.
2.3
Discussion
We have shown that the peel-1/zeel-1 element in C. elegans is composed of two,
tightly linked genes: a sperm-delivered toxin, peel-1, and an embryo-expressed antidote, zeel-1. peel-i and zeel-i are located adjacent to one another in the genome,
and both genes encode transmembrane proteins.
peel-i is expressed in the male
germline, and its product is delivered to the embryo via fibrous body-membranous
organelles. In the absence of zeel-1, sperm-supplied PEEL-1 causes dose-dependent,
late-occurring defects in muscle and epidermal tissue. zeel-i is expressed transiently
in the embryo, and tissue-specific expression of zeel-i produces tissue-specific rescue.
The transmembrane domain of zeel-i is required and partially sufficient for function,
and like peel-1, this domain is evolutionarily novel and does not occur outside C.
elegans. Finally, although PEEL-1 and ZEEL-1 normally function in embryos, peel-i
is lethal when expressed ectopically in adults, and this lethality is rescued by ectopic
expression of zeel-1.
2.3.1
Sperm and early embryos may be protected from PEEL-1
Given the evidence that sperm-supplied PEEL-1 may persist throughout embryogenesis and act directly during the two-fold stage, PEEL-1 must be a remarkably potent
toxin. Sperm are tiny in size compared to the oocyte, roughly 1% as large by volume
[21], so PEEL-1 concentrations in the embryo are necessarily low. Moreover, assum79
ing that PEEL-1 localizes to plasma membranes in the embryo, as might be expected
for a FB-MO protein, then with each cell division, PEEL-1 will become more and
more dilute relative to the total membrane component of the embryo.
Equivalently, the two-fold stage of development must be remarkably sensitive to
the toxic effects of PEEL-1. While the cause of this hypersensitivity remains unclear,
we emphasize that the morphogenetic processes occurring at the two-fold stage involve
changes in cell shape and cell adhesion that are vastly more dramatic than those in
earlier development. In addition, the two tissues most affected by PEEL-1 muscle
and epidermis
are also the two tissues in which these morphogenetic changes are
most pronounced.
The high potency of PEEL-1, combined with its widespread toxicity to a variety
of cell types, highlights an unusual aspect of sperm cell biology: sperm are able to
function normally, despite high concentrations of PEEL-1. While the mechanism of
sperm protection remains unclear, we note that sperm differ from other cell types in
three ways. First, sperm contain only a nucleus, some mitochondria, and FB-MOs;
all other organelles and all ribosomes, are excluded [146, 140]. Second, sperm lack an
actin-based cytoskeleton [93], and instead crawl using polymers of the Major Sperm
Protein [62].
Third, sperm sequester PEEL-1 in FB-MOs.
Such sequestration is
not possible in other cell types because FB-MOs are sperm-specific. In addition, although FB-MOs fuse with the plasma membrane upon sperm activation, they persist
as permanent fusion pores [141]. This morphology prevents at least some FB-MO
proteins from diffusing into the plasma membrane [142], and it may prevent diffusion
of PEEL-1 as well.
The fact that pre-two-fold embryos are able to develop normally even when peel-i
is induced by heat-shock indicates that pre-two-fold development is less sensitive than
the two-fold stage to the toxic effects of PEEL-1. However, given the hypersensitivity of the two-fold stage, it remains unclear whether pre-two-fold embryos are fully
resistant to PEEL-1 (like sperm), or whether PEEL-1 levels in the heat-shocked, pretwo-fold embryos were too low to produce general cytotoxic effects. While we cannot
discount the possibility of full resistance, we note that in the heat-shocked embryos,
80
the time interval between heat-shock and the two-fold stage was five hours, at most.
Five hours was sufficient for necrosis to develop in heat-shocked adults, but the two
heat-shock experiments are not directly comparable because the heat-shock response
in adults and embryos may not be equivalent, and the duration of heat-shock was
shorter in embryos than in adults.
2.3.2
Possible mechanisms of PEEL-1 toxicity and ZEEL-1mediated rescue
Because PEEL-1 has no sequence similarity to any other protein, the PEEL-1 sequence cannot be used to infer the mechanism of its toxicity. The fact that PEEL-1
is toxic even in extremely tiny amounts suggests that PEEL-1 might act catalytically for example, by nucleating aggregation events or by acting as a transmembrane
protease. The muscle hyper-contraction observed in heat-shocked adults, as well as
the paralysis and two-fold arrest observed in peel-i-affected embryos (which may in
theory result from too much muscle contraction rather than too little), suggests that
PEEL-1 might act by releasing intracellular calcium, perhaps by generating a membrane pore. It remains unclear, however, how calcium release alone can account for
the epidermal defects observed in peel-1 -affected embryos, because increased calcium
signaling alone does not cause embryonic arrest [22] and is even known to suppress
certain defects in epidermal morphogenesis [100]. In addition, it remains unclear how
sperm might be protected from increased calcium, given the role of calcium in sperm
activation [142].
Given the uncertain mechanism of PEEL-1 toxicity, there are also many possible mechanisms of ZEEL-1-mediated rescue.
ZEEL-1 might promote degradation
of PEEL-1, or it might prevent PEEL-1 from binding to its target, either by acting as a competitive inhibitor or by neutralizing PEEL-1 through direct interaction.
While we have been unable to demonstrate a direct physical interaction between
PEEL-1 and ZEEL-1, several observations are consistent with it. First, both PEEL-1
and ZEEL-1 are transmembrane proteins. ZEEL-1 localizes to cell membranes, and
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PEEL-i localizes to FB-MOs.
Assuming that FB-MOs do not endocytose during
fertilization, localization to these organelles should deliver PEEL-i to the plasma
membrane of the zygote, where it should have the opportunity to encounter ZEEL1 later in development. Second, the transmembrane domain of ZEEL-1 is required
and partially sufficient for function, consistent with this domain binding directly to
PEEL-1. Third, tissue-specific expression of zeel-1 produces tissue-specific rescue,
consistent with ZEEL-1 being able to neutralize PEEL-i only when both proteins are
present within the same cell. Fourth, ZEEL-I can neutralize PEEL-1 toxicity even
in adults, demonstrating that the genetic interaction between peel-1 and zeel-1 does
not require any intermediaries specific to embryogenesis. Fifth, in both embryos and
adults, ZEEL-i is able to neutralize small but not large doses of PEEL-1. This dosedependence implies that the genetic interaction between peel-1 and zeel-1 requires a
minimum ratio of ZEEL-i to PEEL-1.
2.3.3
Comparison with other genetic elements causing transmission ratio distortion
Like nearly all other selfish genetic elements whose genetic basis is known [30, 79,
80], the peel-1/zeel-1 element experiences a suppression of recombination between
component parts: the insertion/deletion of peel-1 and zeel-1 removes both genes at
once, so the two genes cannot be separated by homologous recombination.
This
genomic organization has undoubtedly allowed peel-i to persist in spite of its toxic
effects, because recombination breaking apart peel-1 and zeel-1 would have generated
haplotypes carrying peel-1 alone, and such haplotypes are effectively suicidal.
The peel-1/zeel-1 elements mode of action is similar to that of Wolbachia, in
that both types of elements act through paternal-effect killing [117, 144]. Wolbachias
molecular mechanism is very different, however, because Wolbachia does not load
sperm with an extra-nuclear toxin, but instead modifies the sperm pronucleus to
undergo a chromatin condensation defect during the first mitotic division [18, 102].
In addition, Wolbachia is an intracellular bacterium, not a nuclear-encoded locus, and
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rescue of Wolbachia-mediatedkilling depends upon the contents of maternal ooplasm,
not zygotic transcription of a nuclear-encoded gene. The peel-1 /zeel-1 elements mode
of action is also similar to the maternal-effect killing and zygotic self-rescue of Medeafactors [11], although the extent of this similarity at the molecular level is unclear
because the mechanism of Medea-factor killing is unknown [79].
2.3.4
Sheltering of the peel-1 /zeel-1
element by near per-
petual homozygosity
Previously, we demonstrated that haplotypes carrying the peel-1 /zeel-1 element and
haplotypes lacking it are maintained by balancing selection [115]. We hypothesized
that the target of selection may be a linked polymorphism, rather than the peel-1 /zeel1 element itself [115].
Under this scenario, peel-1 may represent an unprecedented
case of inverted sheltered load. Sheltered load refers to the incidental maintenance of
deleterious alleles tightly linked to sites under balancing selection [138].
Ordinarily,
sheltered load occurs when deleterious recessives arise on haplotypes maintained in
persistent heterozygosity, such as those of major histocompatibility complex loci in
vertebrates [55] or self-incompatibility loci in plants [125]. peel-1 is like these deleterious recessives in that although it has the potential to impose substantial genetic load
on the species, its effects are rarely visible to natural selection. In the case of peel-1,
however, sheltering is inverted because peel-1 is only visible when heterozygous, and
in C. elegans, heterozygosity is the exception rather than the norm.
Like any locus promoting its own transmission to the detriment of the rest of the
genome, the peel-i/zeel-1 element creates a selective environment favoring its own
suppression.
From a genic perspective, loci unlinked to the peel-1/zeel-i element
suffer a fitness cost every time they are transmitted to a peel-1 -affected embryo. As a
consequence, mutations unlinked to peel-1 and zeel-1 that either suppress the activity
of peel-1 or mimic the activity of zeel-1 will be favored by natural selection. Insofar
as such alleles are accessible in mutational space, their absence further attests to the
sheltering of the peel-1/zeel-i element by near perpetual homozygosity. (The peel-1
83
mutations in strains MY19 and EG4348 do not represent favored alleles because they
do not arise on haplotypes suffering a fitness cost.)
2.3.5
Evolutionary origins of the peel-1 /zeel-1
element
The insertion/deletion polymorphism of peel-1 and zeel-1 raises the following question: Did this indel polymorphism arise by an insertion event or by deletion of preexisting sequence?
With respect to zeel-1, this polymorphism probably arose by
a deletion event, because the divergence between zeel-1 and its presumed ancestor, Y71A12B.17, predates allelic divergence at the peel-1/zeel-1 locus.
zeel-1 and
Y71A 12B. 17 are 45% diverged at the amino acid level, and divergence at synonymous
sites is saturated (see Materials and Methods). In comparison, the Bristol and Hawaii
alleles of genes surrounding the indel polymorphism of peel-1 and zeel-1 are roughly
2% diverged at the amino acid level and 10-16% diverged at synonymous sites [115],
this level of divergence is representative of the divergence between all haplotypes
carrying the peel-1 /zeel-1 element and all haplotypes lacking it [115].
It is reasonable to suppose that the peel-1/zeel-1 element originated as a weak
toxin-antidote pair and then co-evolved into its current form.
Yet given the low
selective pressure for transmission ratio distortion in a self-fertilizing species, it is
unlikely that peel-1 and zeel-1 co-evolved within C. elegans as result of this type of
selective pressure alone. One possible solution to this paradox is that peel-1 and zeel-1
co-evolved in the out-crossing ancestor of C. elegans, where the selective pressure for
transmission ratio distortion would have been much stronger. Another, non-mutually
exclusive hypothesis is that peel-1 was originally favored because it aided in another
cellular process, such as sperm competition, and its toxicity to the embryo was initially
mild and incidental. Under this scenario, zeel-1 would have arisen to counteract the
toxicity of peel-1, and once zeel-1 became established, the presence of zeel-1 would
have allowed for stronger toxicity on the part of peel-1 and, eventually, lethality
in zeel-1s absence. Regardless of the initial selective pressures favoring peel-1 and
zeel-1, however, the fact that both peel-1 and the transmembrane domain of zeel-1
are evolutionarily novel indicates that the self-promoting activity of the peel-1 /zeel-1
84
element arose fundamentally from the co-evolution of two novel proteins.
2.4
Acknowledgements
Foremost, we are very grateful to Michel Labouesse for his generous advice regarding
the analysis of peel-i-affected embryos. We also thank Michael Brauchle for help with
microscopy; Arjun Raj for providing the nuclei counting code; Joshua Bloom for help
with phylogenetic analysis; Andy Singson, Steve LHernault, Christian Frokjr-Jensen,
and Indrani Chatterjee for reagents and protocols; three anonymous reviewers for
comments on the manuscript; and Marie-Anne Flix, Antoine Barrire, Elie Dolgin,
Erik Andersen, the National Bioresource Project of Japan, and the Caenorhabditis
Genetics Center, funded by the NIH National Center for Research Resources, for
strains. MA thanks Erik Jorgensen for allowing him to work on this project in his
laboratory.
85
86
Chapter 3
E- and N-cadherin in the
developing mouse limb
3.1
Introduction
The vertebrate limb is a good model for studying pattern formation and morphogenesis (for review see [150]). The growth of a limb starts with the appearance of a
small protrusion called the limb bud at specific locations of the developing embryo.
The limb bud consists of undifferentiated mesenchymal cells covered by ectoderm.
The epithelial-mesenchymal interactions between the two layers are important for
induction, outgrowth, and patterning of the limb. Several specialized regions called
organizing centers form to coordinate these processes. Among them, the Apical Ectodermal Ridge (AER) is particularly important for the limb outgrowth.
The AER is a thickened layer of ectodermal cells at the tip of a growing limb bud.
The AER is found to be important for limb outgrowth, in that surgical removal of
the AER caused truncation of the limb. Removal of the AER at progressively later
stages results in progressively more distal truncation [95].
The Apical Ectodermal
Cap (AEC), the equivalent structure of AER in amphibians, is the key for triggering
limb regenerative response [113]. The AER is thought to be induced and maintained
by the surrounding ectodermal and underlying mesenchymal cells through Wnt and
FGF signaling, though the specifics are not well-understood [23]. The AER lies at
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the boundary between dorsal and ventral sides of the limb, and this positioning is
controlled by Wnt7a and Eni in mouse [67].
3.1.1
Cadherin switch and Epithelial-mesenchymal transition
(EMT) in development
All cells in our body come from one single cell.
One strategy to enhance cellular
diversity, as an alternative to the differentiation of stem cells, is for differentiated
cells to retain plasticity and to be able to transition back and forth between different
cell types. During development (and adulthood), certain cells in the epithelia have
the ability to convert between epithelial and mesenchymal states via EMT and MET
(Mesenchymal-epithelial transition) [65]. In particular, the transition from epithelial
to mesenchymal state allows increased cell motility and invasiveness.
E-cadherin and N-cadherin are biomarkers of epithelial and mesenchymal cells,
respectively. Therefore, the cadherin switch from E-cadherin to N-cadherin has been
used to monitor the progress of EMT [149]. At the molecular level, EMT transcriptional reprogramming can be activated through common transcription factors such as
Snail, Slug, Twist, ZEB1/2, FOXC [133], which can also be used as markers of EMT
[149].
3.2
Preliminary Results
In this preliminary study, we examined the expression of E-cadherin and N-cadherin
in the forelimb of E9.5 mouse embryos. We have observed E-cadherin to be expressed
exclusively in the limb ectoderm including the AER (Figure 3-1 A). N-cadherin is
expressed in both the underlying limb mesenchyme and the AER but not in the
surrounding ectoderm (Figure 3-1 B). To our surprise, the cadherin expressions in the
AER resemble neither those of epithelial cells undergoing EMT nor those of normal
non-transitioning epithelial cells. In other words, the expression patterns of E- and
N-cadherin are mutually exclusive, except for in the AER, where substantial levels
88
of both cadherins are observed.
Further cell dissociation and sorting experiments
using combined expression levels of E- and N-cadherin as biomarkers can isolate cells
within the AER from those within the ectoderm and mesenchyme layers, and help
further elucidate the molecular mechanisms behind the induction and maintenance
of the AER as well as the epithelial-mesenchymal interactions driving the induction,
outgrowth, and patterning of the limb.
3.2.1
Image stitching method
One big technical difficulty in imaging large mammalian tissue section was customizing automated image stitching software. Commercial and open source packages work
to various degrees. We have adapted a method based on correlation [107] and used
pre-recorded microscope positions for efficient and accurate image registration. The
normalized 2-dimensional(2-D) correlation function measures the similarity between
two input images. The translational offset between two partially overlapping images
can be retrieved by maximizing the 2-D correlation function for each translation. One
efficient way to calculate the normalized 2-D correlation function, using the correlation theorem, is to use fast Fourier transform to calculate the cross-power spectrum
first.
The cross-power spectrum, namely, the Fourier transform of the normalized
2-D correlation function of two input images, is equal to the product of the Fourier
transform of one image and the complex conjugate of the Fourier transform of the
other. By applying the inverse Fourier transform on the cross-power spectrum, one
obtains the normalized 2-D correlation function. To speed up image processing time,
we pre-crop the 1024-pixel-by-1024-pixel images down to a size of 50-pixel-by-50-pixel
based on pre-recorded microscope positions. In theory, the 2-D correlation function
has a single peak, indicating the translational offset between the two input image
patches. But in reality, multiple peaks can result from bright auto-fluorescent spots
in the background and thus become a source of registration error. Pre-cropping reduces the number of bright auto-fluorescent spots down to essentially zero in the
to-be-matched image patches, and therefore drastically improves the accuracy of image stitching. Eighteen 1024-pixel-by-1024-pixel images were stitched together and
89
shown in (Figure 3-1 A).
90
Figure 3-1: E- and N-cadherin in forelimb of mouse E9.5 embryo. E- and Ncadherin mRNAs were visualized in the forelimb of a wild-type, E9.5 mouse embryo
using single-molecule fluorescence in situ hybridization. (A) E-cadherin mRNAs are
shown in white. (B) shows a magnification of the boxed area in (A), with N-cadherin
mRNAs shown in green, and nuclei are stained with DAPI (blue).
91
Appendix A
Supplementary Information
The oligonucleotide sequences of all the probes generated in this study are included
below:
pie-1
atcggctttgtttgagccat, gagtgcagccatttgctcgg, acgacgtgtcatctgagttg, ctgttcgatcgatcggcagc,
cgggcacgtcgcattcagaa, ctactgaattttgaattctc, gatcgattgattttccgttg, ggacgacagcgaatcattga,
cgaagccattttccactata, tcttgagcgcttcacgcttt, tcaatctgagccaatggagt, tcttttagttgccggcgcct,
gettatccttcgccgagcta, agtcgcgttttgtattctgt, ttcacggcggaacgcatcac, tgtcgttgtacgggcagtat,
tgtccgtgagcatatgtgca, tctcggaactctcagctcat, gggaataatactcttggcgg, acggcgctcacgtggtggat, cgtctcgtctagaacgagaa, cgattgattgttgtatccac, cttcgatgctgaagaactcg, gtctccgattctcatcatga, ttcgagcttccgtggttgtt, attgtgacaaatctgacgac, tatctgcagtttcctctctc, aatgaagcggcatcttggac, aatgttgcatttgttcgacg, taaaccgtcgcattcgcatt, acaatcggaagaaggtggcg, tggtagtaggcaatcggcgg, ttgctgatgttgtggatgat, gcattggaaatggcaggaat, ggcggtggagccaagaaata,
aggagctccttgttgagctt, gtggaatatattgcactgga, ggctattcatcaaatcatgt, gccattggtgcatacattgg,
tggttgatagtagtatgtcg, tgggcatgccattcgaatta, cggatcaatagtcacatcca, acttcaaacgcaccgcccgt, agagaagaatccatcgggga, aaataatagttggtggtggc
nos-2
cttggagtacccagagacat, ttcgagagtcgaagtcggtt, aaagatcgtcaaaaatgtct, acatcctctagataatacaa, cggcgttgtggcggaaagga, cactcaaatcaaatgttggc, gaaagtgatggatccgatgg, ccattcact-
93
gtcaaacgaat, ttgagcatggagtcattggt, gttgaggtctccggtctttc, ttcaaacgagaagattgatg, gcggcgttttgatgggtcta, agtcgaagatattcggcagg, attcgttgacaggaatgtcg, ctgaatcgaaaaatccggaa, aaattgaaatccgaagcgac, ccaatcattgaacttcccgt, tgcgaggcttgctgtcattc, gattcaatcgcaaacgatcc, attcctcgcgagctcttcag, gctcggagcgagtttctctt, ttgaacaatgatggcacttc, atccacatccatattcacgc, catatatccaacggaacgac, tacgtgtatgcgtttcccat, gagctgagcttgtcgcattt, cacaaattttacatggagcc, tgtgattcatttcaccgcga, ttcattgggcagtacgtctc, ttgaagaacaactggctcga,
gaagtctcggctgaaatcct, tgcgctggaatcgtcggttt, tgcttgtaaaattgataacg, gatcttctgaattaatgagg, aagaatcttcggatgatgcg
gfp:h2b
agttcttctcctttactcat, aattgggacaactccagtga, catcaccatctaattcaaca, gacagaaaatttgtgcccat, catcaccttcaccctctcca, agggtaagttttccgtatgt, tccagtagtgcaaataaatt, ttggccatggaacaggtagt,
taacagaaagtagtgacaag, tctcgagaagcattgaacac, tgccgtttcatatgatctgg, gcatggcactcttgaaaaag,
ctttcctgtacataaccttc, gtcatctttgaaaaatatag, cagcacgtgtcttgtagttc, gtatcaccttcaaacttgac,
taactcgattctattaacaa, cttctttaaaatcaatacct, ttgtgtccaagaatgtttcc, tgagttatagttgtattcca,
ctgccatgatgtatacattg, ttgattccattcttttgttt, gtgtctaattttgaagttaa, gaacgcttccatcttcaatg,
tgttgataatggtctgctag, gccatcgccaattggagtat, ggttgtctggtaaaaggaca, gcagattgtgtggacaggta,
ttcgttgggatctttcgaaa, gaaggaccatgtggtctctc, ccagcagctgttacaaactc, ttcatccatgccatgtgtaa
peel-1
aaaaccacagttggtgtgac, ggaaatcaaagcgcatcctt, ccgactatgatgttccacaa, caggcagatcaagatacatg, gcaaaccgatttacccatac, gacgagacagaaaatcttcc, gcaaacaagaagcaggatag,
ggc-
gaaaatctgtgcaagaa, catgggtcaatgtaaaggac, gaacagtgcagagttcaatg, gcaccacgagactcataata,
ctaattccatcaacggatgg, ttaaatcgtacagcagcgca, atgagcagttgagttgagac, gccttctacacatcgatcaa
zeel-1
acaactccagtcatttgcct, cagcaaaaaccattgaggtg, cacgcaatgttteaccgtat, cgttggtacaagcatgttgt, cgagcgctataacaagatac, caactagaccaatgaccgta, ggagatacacttttttcgcg, tttcggt-
graaacgtcagaag, gaactggtacagaagcaaca, aaaggaccacatcagactca, ttaaaaaaccccagctggct,
gcaatctgaacacgagtaga, ctcgtcctcagteataacaa, gagcacatcaaggtgagaaa, ccaacccaaattgaaacctc, ccagtgcatgaaccacaata, gctcattctttttgagtgcg, gctgtgttgcaatggttcaa, tgtcattttgtgaggaaggc, cagtgatgtttgccagagaa, catcgttgatatgccttgca, gggtcgtctttgctataaac, ccaattgacagcgacttttc, ctcgcactctctaatgtcta, aacccatcctggagtcaatt, gggaagagagcataatacca, cgcagagttctgagaaatct, cggtgtcgcttatatccaat, ccagcatatcagaggaacat, cagttaggcctgttagtttg, cgcattccacaaattgcatg, ggaagcatctagaaacgtca, ggatgagatgctaaggagtt, gtctgtagattaccgaagct, ctggtggctacagatctttt, gcttctatcgcatgccttaa, ctgctcattgggtctaaaag, gcggatggaatatatgggat,
cgttcggaggcaataagtaa, tgatgatgtaagggggagaa, tactgcttgacaaggatgct, gccagtcaaactcttgatca,
cactggtgccaatttgatca, atagacttccagcgcaaaac, tcaagaaacgatcgggtctt, tctcgtacaggtttccacaa, tccactgaaatcctgacttg, cgctcttactgcataatgtc
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